Succinate prodrug, compositions containing the succinate prodrug and uses thereof

ABSTRACT

The present invention provides a novel isolated succinate prodrug as the free com-pound or a salt, hydrate, solvate or complex thereof being cell permeable and aimed for increasing the ATP-production in mitochondria. The compound is useful in the medical treatment of a range of diseases, in nutritional supplements, nutricosmetics and in cosmetics.

This application is a § 371 application of PCT/EP2020/066923, filed Jun.18, 2020, which in turn claims priority to DK Application PA 201970382,filed Jun. 19, 2019; DK Application PA 201970383, filed Jun. 19, 2019;and DK Application PA 201970384, filed Jun. 19, 2019. The entiredisclosure of each of the foregoing applications is incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to the fields of chemistry,pharmacologically active compounds, pharmaceutical compositionscomprising such compounds and nutrition. Specifically, the inventionrelates to cell-permeable precursors of succinates useful as medicinesand nutritional supplements.

BACKGROUND OF THE INVENTION

Mitochondria are organelles in eukaryotic cells that generate most ofthe cell's supply of adenosine triphosphate (ATP), which is used as anenergy source. Thus, mitochondria are indispensable for energyproduction, for the survival of eukaryotic cells and for correctcellular function. In addition to supplying energy, mitochondria areinvolved in a number of other processes such as redox and ion balance,cell signalling, cellular differentiation, cell death as well as thecontrol of the metabolic processes, cell cycle and cell growth. Inparticular, mitochondria are crucial regulators of cell apoptosis andthey also play a major role in multiple forms of non-apoptotic celldeath such as necrosis.

Mitochondrial dysfunction contributes to a wide variety of diseases andcan be caused by mutations or deletions in the mitochondrial or nucleargenome, primary or secondary impairment of the mitochondrial respiratorysystem or other mechanisms related to abnormal mitochondrial function.At present there is no available treatment that can cure mitochondrialdiseases.

The oxidation of nutrients to produce usable chemical energy in the formof ATP occurs to a large extent in the mitochondria through a series ofchemical reactions in the tricarboxylic acid cycle and the electrontransport chain. NADH generated in the tricarboxylic acid cycle feedsinto complex I in the electron transport chain. Succinate is a metabolicintermediate of the tricarboxylic acid cycle in mitochondria and isunique by being directly metabolized by the enzyme succinatedehydrogenase of complex II in the electron transport chain. Succinatecan also act as a signaling molecule reflecting the cellular metabolicstate.

In view of the recognized importance of maintaining or restoring anormal mitochondrial function or of enhancing the cell's energyproduction (ATP) in the treatment of disease and conditions related tomitochondrial dysfunction or the enhancement of mitochondrial function,there is a need for compounds having cell permeability, the ability toliberate intracellular succinate or a precursor of succinate, lowtoxicity of the compound and intracellularly released by-products, andphysicochemical properties consistent with administration to a subjector patient.

Succinate compounds have been prepared as prodrugs of other activeagents, for example WO 2002/28345 describes succinic acid bis(2,2-dimethyl propionyloxymethyl) ester, succinic aciddibutyryloxymethyl ester and succinic acid bis-(1-butyryloxyethyl)ester.These compounds as prepared deliver formaldehyde, and are aimed atdifferent medical uses compared to the current compounds.

Various succinate ester compounds are known in the art.

WO97/47584 discloses polyol succinates comprising multiple succinatemoieties linked together.

WO2015/155231 discloses succinates and precursors of succinate which arecell permeable.

Murli et al. (Appl. Environ. Microbiol. 71:2005:4503-4509) discloses theattempted chemobiosynthesis of 6-deoxyerythronolide B analogues byfeeding the bacteria Escherichia coli and Streptomyces coelicoloracyl-thioesters. A table of structures discloses various acyl-thioestersincluding the formal structure of methyl3-[(2-acetylaminoethylthio)carbonyl]propionate but synthesis failed anddid not give the desired product.

There is a need for effective and safe new treatment options fordiseases having their origin in mitochondrial dysfunction or forenhancing metabolism by delivering a substrate of metabolism. There isalso a need for new nutritional supplements, nutricosmetics,cosmeceuticals and cosmetics for stimulating energy in a subject and forserving as antioxidant. Such new treatments, nutritional supplements,nutricosmetics, cosmeceuticals and cosmetics are required to haveattractive combinations of properties including high activity to enhancemitochondrial energy production and or to serve as anti-oxidant, goodbioavailability, long plasma half-life, stability when formulated into aproduct and low toxicity. In particular there is a need for such newtreatments, nutritional supplements, nutricosmetics cosmeceuticals andcosmetics that are based on an active ingredient having a highsolubility in water, good cell permeability and when desirable a highblood brain barrier penetration and/or gives reduced lactate production.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides isolated Methyl3-[(2-acetylaminoethylthio)carbonyl]propionate (Compound 1) in solidform. It may be in the free form or a salt, hydrate, solvate or complexthereof.

Compound 1 has the structure (formula 1):

It has now surprisingly been found that the cell permeable Compound 1has a remarkable combination of advantageous properties. It has potentin vivo activity of stimulating the energy production in mitochondriaand has good oral bioavailability, blood brain barrier penetration, goodplasma stability, and decrease lactate production and restores succinatelevels. Simultaneously, Compound 1 possesses remarkably high solubilityin water and aqueous systems. Solubility assessment has shownsolubilities in excess of 500 mg/mL, which equates to ˜2.1 M. Thisextremely high aqueous solubility likely comes from the low meltingpoint of Compound 1 (less than 55° C.), and on addition of aqueoussolvent it is miscible with the aqueous solvent. This enables very highconcentration of aqueous formulations, allowing for high oral dosing ofa compound which is in effect a prodrug of a substrate for metabolism.

In an embodiment the isolated Compound 1 is a solid product with amelting point or melting range in the range of from about 35° C. toabout 55° C. In preferred embodiments the isolated Compound 1 has apurity of at least 80% w/w, such as at least 85% w/w, at least 90% w/w,such as at least 95% w/w, but it may also have a lower purity such as atleast 30% w/w, at least 40% w/w, at least 45% ww, at least 50% w/w, atleast 55% w/w, at least 60% w/w, at least 65% w/w, at least 70% w/w orat least 75% w/w. Dependent on the manufacturing method and the storageconditions, Compound 1 may comprise crystals and/or it may comprisenon-crystals such as amorphous forms of Compound 1, and mixturesthereof. As seen from the Examples herein, all methods used lead toCompound 1 with a degree of crystallinity.

It is contemplated that more than one crystal form of Compound 1 mayexist and Compound 1 may also exist as an amorphous solid’. In thepresent context, all forms of Compound 1 is within the scope of thepresent application including mixtures of two of more forms ofCompound 1. Thus, the term “Compound 1” denotes a compound of formula 1in solid form, but irrespective of whether the compound is in acrystalline form, in an amorphous form, in a polymorphous form in powderform or in mixtures thereof.

In particular, it has been found that Compound 1 has a number of solidforms with differing properties. For example, as an amorphous solid oras a mainly amorphous solid, it shows higher kinetic solubility. It alsohas crystalline forms (or mainly crystalline forms) which have beengenerated and show other improved properties when handled as a solidform. It has also been found that the purity affects the properties ofthe preparation. In particular, the melting point is low and close tobody temperature and is altered by the presence of impurities. Thestability of Compound 1 preparations is also altered by the presence ofimpurities, such as those in non-purified water, which reduce thestability of Compound 1.

In a second aspect the present invention provides a compositioncomprising isolated Compound 1.

In a third aspect the present invention provides a cosmeceuticalcomprising the isolated Compound 1.

In a fourth aspect the present invention provides a nutricosmeticscomprising the isolated Compound 1.

In a fifth aspect the present invention provides a process for preparingisolated Compound 1, said process comprising the steps of:

a) reacting N-acetyl cysteamine and monomethyl succinate, in thepresence of a coupling reagent, in organic solvent, between 0° C. and100° C.

b) isolating Compound 1, so as to provide isolated Compound 1.

The method normally includes a purification step to increase the purityof the compound.

The Compound 1 compounds of the invention can be used to enhance orrestore energy production in mitochondria. Notably the compounds can beused in medicine, nutricosmetics, nutritional supplements,cosmeceuticals and in cosmetics. The Compound 1 compounds can be used inthe prevention or treatment of disorders or diseases having a componentrelating to mitochondrial dysfunction and/or to a component of energy(ATP) deficiency as well as to utilize the cell signalling properties ofsuccinate and its anaplerotic effects on metabolic intermediates.

In addition, in comparison with known succinate prodrugs (such as e.g.mentioned in WO 97/47584), the isolated Compound 1 of the presentinvention shows improved properties for treatment and for use asnutritional supplement and cosmetic product, including better cellpermeability, longer plasma half-life, good oral bioavailability,reduced toxicity, increased energy release to mitochondria, and improvedformulation properties e.g. due to improved solubility in water.

In another aspect, the invention provides a pharmaceutical compositioncomprising Compound 1 compounds.

The pharmaceutical composition may be a solid formulation or it may be asolid formulation for reconstitution prior to use.

Alternatively, it may be in the form of a liquid such a an aqueoussolution including e.g. an aqueous Phosphate Buffered Saline (PBS)formulation. In general, a pharmaceutical composition of the inventionhas a concentration of Compound 1 of at least 10% w/w, at least 30% w/w,at least 50% w/w, at least 60% or at least 70% w/w. In an embodiment,the pharmaceutical composition is a solution of Compound 1 in purifiedwater optionally made isotonic with the blood.

In another aspect the present invention provides the use of Compound 1of a composition thereof in the treatment or prevention of a metabolicdisease, a disease of mitochondrial dysfunction, a disease related tomitochondrial dysfunction, a mitochondrial disorder, mitochondrialenergy deficiency, drug-induced mitochondrial side effects, cancer,diabetes, traumatic brain injury, acute liver injury and atrialfibrillation.

In an aspect the present invention provides a process for preparing apharmaceutical composition comprising Compound 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. LCMS analysis of Compound 1 Batch 3 depicting Absorbance Unit(AU) versus time using HPLC Method 2.

FIG. 2. LCMS analysis of Compound 1 Batch 3 depicting total ion count ona normalised scale versus time using HPLC Method 2.

FIG. 3. LCMS analysis of Compound 1 Batch 3 depicting intensity (%)versus m/z using HPLC Method 2.

FIG. 4. LCMS analysis of Compound 1 Batch 12 depicting Absorbance Units(AU) versus time using HPLC Method 2.

FIG. 5. LCMS analysis of Compound 1 Batch 12 depicting total ion counton a normalised scale versus time using HPLC Method 2.

FIG. 6. LCMS analysis of Compound 1 Batch 12 depicting intensity (%)versus m/z using HPLC Method 2.

FIG. 7A-B. Intravenous infusion of PBS or Compound 1 in an anesthesisedpig depicting succinate concentration in plasma versus time of infusionin (A), and fumarate concentrations in tissue in (B).

FIG. 7C. Intravenous infusion of PBS or Compound 1 in an anesthesisedpig depicting effect on blood lactate concentrations.

FIG. 8. Intravenous infusion of PBS or Compound 1 in an anesthesised pigsimultaneously infused with the complex 1 inhibitor rotenone, depictingsuccinate concentrations in tissues at the end of infusion in (A), andlactate concentrations in brain microdialysates expressed as percent ofthe baseline value before initiation of infusion versus time in (B).

FIG. 9. Treatment of Ndufs4 KO mice with Compound 1 in the drinkingwater depicting body weight development versus time in (A) and percentsurvival versus time in (B).

FIG. 10. Treatment of rotenone injected rats with Compound 1 in thedrinking water depicting number of rearings versus treatment in (A),displacement distance in a postural instability test versus treatment in(B) and blood lactate concentration versus treatment in (C).

FIG. 11. XRPD analysis of Compound 1 Batch 12

FIG. 12. XRPD analysis of Compound 1 Batch 15

FIG. 13. LCMS analysis of Compound 1 Batch 3 depicting Absorbance Unit(AU) versus time using HPLC Method 1.

FIG. 14. LCMS analysis of Compound 1 Batch 3 depicting total ion counton a normalised scale versus time using HPLC Method 1.

FIG. 15. LCMS analysis of Compound 1 Batch 3 depicting intensity (%)versus m/z using HPLC Method 1.

FIG. 16. LCMS analysis of Compound 1 Batch 12 depicting Absorbance Units(AU) versus time using HPLC Method 1.

FIG. 17. LCMS analysis of Compound 1 Batch 12 depicting total ion counton a normalised scale versus time using HPLC Method 1.

FIG. 18. LCMS analysis of Compound 1 Batch 12 depicting intensity (%)versus m/z using HPLC Method 1.

FIG. 19. XRPD analysis of Compound 1 Batch 3.

FIG. 20. XRPD analysis of Compound 1 Batch 18.

FIG. 21. XRPD analysis of Compound 1 Batch 13.

FIG. 22. XRPD analysis of Compound 1 Batch 14.

FIG. 23. XRPD analysis of Compound 1 Batch 19.

FIG. 24. XRPD analysis of Compound 1 Batch 16.

FIG. 25. XRPD analysis of Compound 1 Batch 17.

DESCRIPTION OF THE INVENTION

In a first aspect the present invention provides isolated Methyl3-[(2-acetylaminoethylthio)carbonyl]propionate (Compound 1) in solidfrom. It may be in the free form or a salt, hydrate, solvate or complexthereof.

Compound 1 has the structure (formula 1):

As mentioned above, Compound 1 may be in the form of a salt. Suitablesalts include pharmaceutically acceptable salts such as hydrochloridesalt, hydrobromide salt, acetate, citrate, lactate, maleate, malonate orthe like.

Compound 1 may also be a solvate. Suitable solvates may includehydrates, ethanolates

Compound 1 may also be in the form of a complex. Examples of suitablecomplexes may be Compound 1 complexed with cyclodextrin, lipids,triglycerides, carbohydrates, PVA,

In an embodiment the isolated Compound 1 is a solid product with amelting point or melting range in the range of from about 35° C. toabout 55° C. As seen from the examples herein Compound 1 has beenprovided with different melting points most likely dependent on thecontent of different forms of Compound 1 such as crystal forms,amorphous forms etc. In particular, melting points in the range of from39 to 51° C. have been found such as melting points of 39° C. andmelting points in the range of from 46 to 51° C. such as about 46-47°C., 48-49° C. and 50-51° C.

In preferred embodiments the isolated Compound 1 has a purity of atleast 80% w/w, such as at least 85% w/w, at least 90% w/w, such as atleast 95% w/w or at least 97% w/w, but it may also have a lower puritysuch as at least 30% w/w, at least 40% w/w, at least 45% ww, at least50% w/w, at least 55% w/w, at least 60% w/w, at least 65% w/w, at least70% w/w or at least 75% w/w. Dependent on the manufacturing method andthe storage conditions, Compound 1 may comprise crystals and/or it maycomprise non-crystals such as amorphous forms of Compound 1, andmixtures thereof. As seen from the Examples herein, all methods usedlead to Compound 1 with a degree of crystallinity. Compound 1 may alsoappear as a powder.

As seen from the Examples herein, Compound 1 has an excellent watersolubility at room temperature (20-25° C.). At pH 7.4 and in the aqueousmedia tested in the examples, Compound 1 has an aqueous solubility of atleast 300 mg/mL. The aqueous solubility of Compound 1 is dependent onthe crystallinity of the Compound; thus the lower degree ofcrystallinity, the higher aqueous solubility. As seen from Example 10herein a mainly amorphous material may have an aqueous solubility of 850mg/mL. It is therefore contemplated that the aqueous solubility ofCompound 1 is in a range of from 300 mg/mL to about 900 mg/mL.

The kinetic solubility has also been determined and the rate constantsfor the kinetic solubility have been found to be in a range of from0.005 to 0.2 s⁻¹, such as in a range of from 0.01 to 0.15 s⁻¹. Thekinetic solubility may depend on various factors such as particle size,crystallinity, content of amorphous material etc.

Regarding the crystallinity of Compound 1 it may have a degree ofcrystallinity in a range of from 0% to 100% such as from 10% to 100%,from 20% ti 100%, from 30% to 100%, from 40% to 100%, from 50% to 100%,from 60% to 100%. As seen from the Examples herein many of the batchesprepared by the method described herein have a crystallinity of at least50% such as in a range of from about 50% to about 80%.

As seen from the XRPD data in the examples, crystals of Compound 1 arecharacterized by having an X-ray powder diffraction pattern with signalsat 21.4, 22.2, 22.8, 23.1 and 23.3 (±0.2 degrees, 2-theta values).

Crystals of Compound 1 may also have one or more signals at 10.9, 13.1,14.9, 16.2, 20.1, 24.0, 24.8, 26.1, such as two or more, three or more,four or more, six or more, seven or more, or eight. As seen from theexamples, almost all the tested compounds have signals at these degrees(±0.2 degrees, 2-theta values).

From the data in the Examples it is contemplated that signals at 11.1and 16.9 (±0.2 degrees, 2-theta values) relate to a polymorphous form ofCompound 1 (Form 1). Thus, crystals of Compound 1 may have an X-raypowder diffraction pattern with signals at 11.1 and 16.9 (±0.2 degrees,2-theta values), either in complement to one or more of the signalsmentioned above, or in the alternative.

As mentioned above, Compound 1 is in solid form notably comprisingcrystals of the compound. The melting point is rather low, but it isadvantageous that Compound 1 is not in the form of an oil. First of all,it will be easier to handle Compound 1 in manufacturing ofpharmaceutical/cosmeceutical composition (eg millability, bulk powderflow and compressibility). Secondly, the crystalline form is normallythe most stable form and noncrystalline (less ordered) material tends tochange form to crystalline (more ordered, lower energy) over time.

Definitions

The term “Compound 1” denotes a compound of formula 1 in solid form andthe term includes all crystalline forms, all amorphous forms, allpolymorphous forms, and mixtures thereof including mixtures within thesame form or within different forms. Compound 1 may also be in powderform.

The term “purity” as used herein in relation to Compound 1 means thedegree to which a Compound 1 composition is methyl3-[(2-acetylaminoethylthio)carbonyl]propionate (Compound 1) relative tothe total of Compound 1 and the related impurities being by-products,aberrant forms of Compound 1 (closely related structure) and synthesisprecursors for Compound 1. Hence, in a composition containing 10% w/wCompound 1, the purity of said Compound 1 may be e.g. 95% w/w or 50%w/w, meaning that the Compound 1 used to make said composition has apurity of 95% w/w or 50% w/w, respectively. Purity can be assessed byone of a number of methods including qNMR, HPLC etc. In qNMR a knownamount of analyte is dissolved in NMR solvent with a known amount ofinternal standard. A ¹H NMR spectrum is obtained, with sufficient scansto reduce the signal to noise ratio. An exemplary resonance in theinternal standard and the analyte are integrated. The ratio of theseintegrals, coupled with the knowledge of how many protons the signal iscomprised and the molecular weights of both analyte and internalstandard, is then used to determine the purity in a w/w %. In HPLC thepurity is assessed as the area under curve (AUC) for the analyte incomparison to other signals with different retention times.

The term “isolated” as used herein in relation to Compound 1 means theCompound 1 product methyl 3-[(2-acetylaminoethylthio)carbonyl]propionateas obtained from a synthesis reaction and isolated e.g. by purificationfrom various by-products, synthesis precursors and aberrant Compound 1forms.

The term ‘nutricosmetics’ as used herein refers to nutritionalsupplements or cosmetics specifically formulated to help maintainhealthy skin, hair and nails with active ingredients that supportphysiological functions to achieve a healthier and more youthfulappearance over time. Unlike a topical cream or treatment,nutricosmetics are taken orally and work from the inside to promotehealthy skin, hair or nails from within.

The term “cosmeceutical” as used herein is intended to mean a cosmeticproduct with bioactive ingredients purported to have medical benefits.Cosmeceutical products are marketed as cosmetics, but reputedly containat least one biologically active ingredient. Examples of cosmeceuticalsinclude anti-wrinkle skin creams with ingredients such as alpha lipoicacid and dimethylaminoethanol and creams containing “cellularreplenishment serum” that are stated as having “antiaging properties”.

The term “treatment” as used herein is intended to mean implementationof therapy with the intention of reducing the severity or frequency ofsymptoms. As used herein the term “treatment” refers to both therapeutictreatment and prophylactic or preventive measures.

The term “prevention” as used herein is intended to mean preventing inwhole or in part, or ameliorating, reducing or controlling.

Compound 1 is generically covered by the Formula (I) of WO2015/155231disclosing succinates and precursors of succinate which are cellpermeable. However, with the identification of Compound I the inventorsmade a number of new surprising discoveries revealing that it hasunexpected good combination of properties that make it suitable for anumber of therapeutic and non-therapeutic uses. Additionally, surprisingdiscoveries have been made around the advantages of certain forms andformulations of Compound I.

General Use of the Compounds of the Invention

Methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate (Compound 1) beingthe free form or a salt, hydrate, solvate or complex thereof asdescribed herein can be used in medicine, notably in the medicaltreatment or prevention of a mitochondria-related condition, disease ordisorder, in nutricosmetics or in cosmetics. Compound I can also be usedin the manufacture of a composition for such medical treatment, orprevention, nutricosmetics or cosmetics. The Compound 1 or a salt,hydrate, solvate or a complex thereof can be used in any situation wherean enhanced or restored energy production (ATP) is desired, such as inthe medical treatment of a disease. The medical treatment may be ofmetabolic diseases, or in the treatment of diseases or conditions ofmitochondrial dysfunction or diseases associated with reduced levels ofsuccinate or functional activity of succinate, or disease where theanaplerotic effect of succinate or its signaling properties are useful,treating or suppressing mitochondrial disorders. The Compound 1compounds may be used in the stimulation of mitochondrial energyproduction and in the restoration of drug- or chemically inducedmitochondrial dysfunction such as e.g. sensineural hearing loss ortinnitus (side effect of certain antibiotics due to mitochondrialtoxicity), poisoning with chemicals or gasses affecting mitochondrialmetabolism, or lactic acidosis. The compounds may be used in thetreatment of cancer, diabetes, acute starvation, endotoxemia, sepsis,systemic inflammatory response syndrome, multiple organ dysfunctionsyndrome and following hypoxia, ischemia, stroke, myocardial infarction,acute angina, an acute kidney injury, coronary occlusion and atrialfibrillation, or to avoid or counteract reperfusion injuries. Moreover,it is envisaged that the compounds of the invention may be beneficial intreatment of male infertility and menopausal symptoms in women.

It is envisaged that the Compound 1 compounds of the invention willprovide cell-permeable precursors of components of the Kreb's cycle andoptionally glycolysis pathways. It is envisaged that following entryinto the cell, enzymatic or chemical hydrolysis will liberate succinate.This hydrolysis of Compound 1 is also regarded as especiallyadvantageous as the thiol group released has reductive properties. Manydiseases have an unwanted oxidative stress component, which may lead todamage to cell structure and cell function. Also, oxidative stress isbelieved to be involved in ageing processes. Accordingly, release of acomponent which can act as an antioxidant and scavenge free radicals orreduce oxygen-reactive species is expected to give extra benefit in bothmedical, nutricosmetic and cosmetic use.

Compound 1 can be used to enhance or restore energy production inmitochondria. Compound 1 can also be used as an antioxidant and scavengefree radicals or reduce oxygen-reactive species. Compound 1 can be usedin the prevention or treatment of disorders or diseases having acomponent relating to mitochondrial dysfunction and/or to a component ofenergy (ATP) deficiency, as well as diseases associated with reducedlevels of succinate or functional activity of succinate, or diseaseswhere the anaplerotic effect of succinate or its signaling propertiesare useful.

Enhancement of energy production is e.g. relevant in subjects sufferingfrom a mitochondrial defect, disorder or disease. Mitochondrial diseasesresult from dysfunction of the mitochondria, which are specializedcompartments present in every cell of the body except red blood cells.When mitochondrial function decreases, the energy generated within thecell is reduced and cell injury or cell death will follow.

Diseases of the mitochondria appear most often in organs that are veryenergy demanding such as retina, the cochlea, the brain, heart, liver,skeletal muscles, kidney and the endocrine and respiratory system.Symptoms of a mitochondrial disease may include loss of motor control,muscle weakness and pain, seizures, visual/hearing problems, cardiacdiseases, liver diseases, gastrointestinal disorders, swallowingdifficulties, fatigue and more. A mitochondrial disease may be inheritedor may be due to spontaneous mutations, which lead to altered functionsof the proteins or RNA molecules normally residing in the mitochondria.Many diseases have been found to involve a mitochondrial deficiency suchas a Complex I, II, III or IV deficiency or an enzyme deficiency likee.g. pyruvate dehydrogenase deficiency. However, the picture is complex,and many factors may be involved in the diseases.

Up to now, no curative treatments are available. The only treatmentsavailable are such that can alleviate the symptoms and delay theprogression of the disease.

Accordingly, the findings by the present inventors and described hereinare very important as they demonstrate the beneficial effect of the cellpermeable Compound 1 being a thioester prodrug of succinic acid on theenergy production in the mitochondria.

In addition, in comparison with known succinate prodrugs (such as e.g.mentioned in WO 97/47584), the isolated Compound 1 compounds of thepresent invention show improved properties for medical treatment and useas nutricosmetics, nutritional supplement, cosmeceutical and cosmeticproduct, including better cell permeability, longer plasma half-life,reduced toxicity, increased energy release to mitochondria, and improvedformulation (due to improved properties including increased solubility).In some cases, the isolated Compound 1 compounds are also orallybioavailable, which allows for easier administration.

Thus, the advantageous properties of the isolated compound of theinvention may include one or more of the following:

-   -   Increased cell permeability    -   Increased oral bioavailability    -   Longer half-life in plasma    -   Reduced toxicity    -   Increased energy release to mitochondria    -   Increased antioxidant activity    -   Improved formulation    -   Increased solubility

The present invention provides Compound 1 for use as in medicine, as apharmaceutically active substance, in particular in the treatment ofcellular energy (ATP)-deficiency.

A compound of the invention may be used in the treatment of complex Iimpairment, either dysfunction of the complex itself or any condition ordisease that limits the supply of NADH to Complex I, e.g. dysfunction ofKrebs cycle, glycolysis, beta-oxidation, pyruvate metabolism and eventransport of glucose or Complex-I-related substrates.

The present invention also provides a method of treatment ofmitochondrial complex I related disorders such as but not limited to,Leigh Syndrome, Leber's hereditary optic neuropathy (LHON), MELAS(mitochondrial encephalomyopathy, lactic acidosis, and stroke-likeepisodes), mitochondrial deletion syndromes, mitochondrial myopathiesand MERRF (myoclonic epilepsy with ragged red fibers), which comprisesadministering to a subject in need thereof an effective amount of thecompound of the invention.

The present invention also provides the use of the compounds of theinvention for the manufacture of a medicament for the treatment oftoxin- or drug-induced lactic acidosis/mitochondrial dysfunction.

Isolated Compound 1 may also be useful in any condition where extraenergy production would potentially be beneficial such as, but notlimited to, prolonged surgery and intensive care.

Mitochondria

Mitochondria are organelles in eukaryotic cells, popularly referred toas the “powerhouse” of the cell. One of their primary functions isoxidative phosphorylation. The molecule adenosine triphosphate (ATP)functions as an energy “currency” or energy carrier in the cell, andeukaryotic cells derive the majority of their ATP from biochemicalprocesses carried out by mitochondria. These biochemical processesinclude the citric acid cycle (the tricarboxylic acid cycle, or Kreb'scycle), which generates reduced nicotinamide adenine dinucleotide (NADH)from oxidized nicotinamide adenine dinucleotide (NAD+) and reducedflavin adenine dinucleotide (FADH2) from oxidized flavin adeninedinucleotide (FAD), as well as oxidative phosphorylation, during whichNADH and FADH2 is oxidized back to NAD+ and FAD.

The electrons released by oxidation of NADH are shuttled down a seriesof protein complexes (Complex I, Complex II, Complex III, and ComplexIV) known as the electron transport chain or the respiratory chain. Theoxidation of succinate occurs at Complex II (succinate dehydrogenasecomplex) and FAD is a prosthetic group in the enzyme complex succinatedehydrogenase (complex II). The respiratory complexes are embedded inthe inner membrane of the mitochondrion. Complex IV, at the end of thechain, transfers the electrons to oxygen, which is reduced to water. Theenergy released as these electrons traverse the complexes is used togenerate a proton gradient across the inner membrane of themitochondrion, which creates an electrochemical potential across theinner membrane. Another protein complex, Complex V (which is notdirectly associated with Complexes I, II, III and IV) uses the energystored by the electrochemical gradient to convert ADP into ATP.

The citric/tricarboxylic acid cycle and oxidative phosphorylation arepreceded by glycolysis, in which a molecule of glucose is broken downinto two molecules of pyruvate, with net generation of two molecules ofATP per molecule of glucose. The pyruvate molecules then enter themitochondria, where they are completely oxidized to CO₂ and H₂O viaoxidative phosphorylation (the overall process is known as aerobicrespiration). The complete oxidation of the two pyruvate molecules tocarbon dioxide and water yields about at least 28-29 molecules of ATP,in addition to the 2 molecules of ATP generated by transforming glucoseinto two pyruvate molecules. If oxygen is not available, the pyruvatemolecule does not enter the mitochondria, but rather is converted tolactate, in the process of anaerobic respiration.

The overall net yield per molecule of glucose is thus approximately atleast 30-31 ATP molecules. ATP is used to power, directly or indirectly,almost every other biochemical reaction in the cell. Thus, the extra(approximately) at least 28 or 29 molecules of ATP contributed byoxidative phosphorylation during aerobic respiration are critical to theproper functioning of the cell. Lack of oxygen prevents aerobicrespiration and will result in eventual death of almost all aerobicorganisms; a few organisms, such as yeast, are able to survive usingeither aerobic or anaerobic respiration.

When cells in an organism are temporarily deprived of oxygen, anaerobicrespiration is utilized until oxygen again becomes available or the celldies. The pyruvate generated during glycolysis is converted to lactateduring anaerobic respiration. The build-up of lactic acid is believed tobe responsible for muscle fatigue during intense periods of activity,when oxygen cannot be supplied to the muscle cells. When oxygen againbecomes available, the lactate is converted back into pyruvate for usein oxidative phosphorylation.

Mitochondrial dysfunction contributes to various disease states. Somemitochondrial diseases are due to mutations or deletions in themitochondrial genome or nuclear. If a threshold proportion ofmitochondria in the cell are defective, and if a threshold proportion ofsuch cells within a tissue have defective mitochondria, symptoms oftissue or organ dysfunction can result. Practically any tissue can beaffected, and a large variety of symptoms may be present, depending onthe extent to which different tissues are involved.

Use of the Compound of the Invention

The compound of the invention may be used in any situation where anenhanced or restored energy production (ATP) is desired. Examples aree.g. in all clinical conditions where there is a potential benefit ofincreased mitochondrial ATP-production or a restoration of mitochondrialfunction, such as in the restoration of drug- or chemically inducedmitochondrial dysfunction or lactic acidosis conditions associated withreduced levels of succinate or functional activity of succinate,conditions where the anaplerotic effect of succinate or its signalingproperties are useful, and the treatment of inborn errors of metabolism,cancer, diabetes, acute starvation, endotoxemia, sepsis, reduced hearingvisual acuity, systemic inflammatory response syndrome and multipleorgan dysfunction syndrome.

In particular, Compound 1 can be used in medicine, notably in thetreatment or prevention of a mitochondria-related condition, disease ordisorder, in nutricosmetics or in cosmetics.

Dysfunction of mitochondria is also described in relation to renaltubular acidosis; motor neuron diseases; other neurological diseases;epilepsy; genetic diseases; Huntington's Disease; mood disorders;schizophrenia; bipolar disorder; age-associated diseases; cerebralvascular accidents, macular degeneration; diabetes; menopausal symptomsand cancer.

Compound 1 for Use in Mitochondrial Related Disorders or Diseases

The compound according to the invention may be used in the prevention ortreatment a mitochondria-related disease selected from the following:

-   -   Aging    -   Alpers Disease (Progressive Infantile Poliodystrophy),    -   Alzheimer's disease    -   Amyotrophic lateral sclerosis (ALS),    -   Autism,    -   Barth syndrome (Lethal Infantile Cardiomyopathy),    -   Beta-oxidation Defects, Bioenergetic metabolism deficiency,    -   Carnitine-Acyl-Carnitine Deficiency,    -   Carnitine Deficiency,    -   Creatine Deficiency Syndromes (Cerebral Creatine Deficiency        Syndromes (CCDS) includes: Guanidinoaceteate Methyltransferase        Deficiency (GAMT Deficiency), L-Arginine: Glycine        Amidinotransferase Deficiency (AGAT Deficiency), and        SLC6A8-Related Creatine Transporter Deficiency (SLC6A8        Deficiency),    -   Co-Enzyme Q10 Deficiency,    -   Complex I Deficiency (NADH dehydrogenase (NADH-CoQ reductase        deficiency),    -   Complex II Deficiency (Succinate dehydrogenase deficiency),    -   Complex III Deficiency (Ubiquinone-cytochrome c oxidoreductase        deficiency),    -   Complex IV Deficiency/COX Deficiency (Cytochrome c oxidase        deficiency is caused by a defect in Complex IV of the        respiratory chain),    -   Complex V Deficiency (ATP synthase deficiency),    -   COX Deficiency, CPEO (Chronic Progressive External        Ophthalmoplegia Syndrome), CPT I Deficiency,    -   CPT II Deficiency,    -   Diabetes type II,    -   Friedreich's ataxia (FRDA or FA),    -   Glutaric Aciduria Type II,    -   KSS (Kearns-Sayre Syndrome),    -   Lactic Acidosis,    -   LCAD (Long-Chain Acyl-CoA Dehydrogenase Deficiency),    -   LC-FAOD (Long-Chain Fatty Acid Oxidation Disorders)    -   LCHAD, Leigh Disease or Syndrome (Subacute Necrotizing        Encephalomyelopathy),    -   LHON (Leber's hereditary optic neuropathy),    -   Luft Disease,    -   MCAD (Medium-Chain Acyl-CoA Dehydrogenase Deficiency),    -   MELAS (Mitochondrial Encephalomyopathy Lactic Acidosis and        Strokelike Episodes),    -   MERRF (Myoclonic Epilepsy and Ragged-Red Fiber Disease),    -   METHYLMALONYL-CoA EPIMERASE DEFICIENCY,    -   METHYLMALONYL-CoA MUTASE DEFICIENCY,    -   MITOCHONDRIAL DNA DEPLETION SYNDROME 5,    -   MITOCHONDRIAL DNA DEPLETION SYNDROME 9,    -   MITOCHONDRIAL DNA DEPLETION SYNDROME 15 (HEPATOCEREBRAL TYPE) (1        family),    -   Maternally inherited diabetes and deafness,    -   MIRAS (Mitochondrial Recessive Ataxia Syndrome),    -   Mitochondrial Cytopathy,    -   Mitochondrial DNA Depletion,    -   Mitochondrial Encephalopathy including: Encephalomyopathy and        Encephalomyelopathy, Mitochondrial Myopathy,    -   MNGIE (Myoneurogastointestinal Disorder and Encephalopathy,    -   NARP (Neuropathy, Ataxia, and Retinitis Pigmentosa),    -   Neurodegenerative disorders associated with Parkinson's,        Alzheimer's or Huntington's disease,    -   Parkinson's disease    -   Pearson Syndrome,    -   Progressive external ophtalmoplegia,    -   Propionic academia,    -   Pyruvate Dehydrogenase Deficiency,    -   POLG Mutations,    -   Respiratory Chain Deficiencies,    -   SCAD (Short-Chain Acyl-CoA Dehydrogenase Deficiency),    -   SCHAD and    -   VLCAD (Very Long-Chain Acyl-CoA Dehydrogenase Deficiency).

Of specific interest is the use of Compound 1 in the treatment of LeighSyndrome, LHON, MELAS, MERRF (myoclonic epilepsy with ragged redfibers).and other diseases/conditions relating to Complex I defects.

Use of Compounds of the Invention in Cosmetics

The compounds according to the invention may be used in Cosmetics forthe following:

-   -   Improved metabolic function in dermal cells (aging skin)    -   Astringent (acne)

Use of Compounds of the Invention as Nutritional Supplements

The compounds according to the invention may be used as nutritionalsupplements for the following:

-   -   Increased energy demand due to strenuous physical activity    -   Increased energy demand due to metabolic decompensation during        infections and surgery    -   Enhanced muscle recovery via rapid distribution to tissue and        by-passing glycolysis

Pharmaceutical Compositions Comprising a Compound of the Invention

The present invention also provides a pharmaceutical compositioncomprising the isolated Compound 1 compounds of the invention togetherwith one or more pharmaceutically acceptable diluents or carriers.

The compound of the invention or a formulation thereof may beadministered by any conventional method for example but withoutlimitation it may be administered parenterally, orally, topically(including mucosal, buccal, sublingual. transdermal or to the skin), viaa medical device (e.g. a stent), by inhalation or via injection orinfusion (intraveneous, subcutaneous, intramuscular etc.). The treatmentmay consist of a single dose or a plurality of doses over a period oftime.

The treatment may be by administration once daily, twice daily, threetimes daily, four times daily etc. The treatment may also be bycontinuous administration such as e.g. administration intravenous bydrop.

Whilst it is possible for the compound of the invention to beadministered alone, it is preferable to present it as a pharmaceuticalformulation, together with one or more acceptable carriers. Thecarrier(s) must be “acceptable” in the sense of being compatible withthe compound of the invention and not deleterious to the recipientsthereof. Examples of suitable carriers are described in more detailbelow.

The formulations may conveniently be presented in dosage form such as aunit dosage form and may be prepared by any of the methods well known inthe art of pharmacy. Such methods include the step of bringing intoassociation the active ingredient (compound of the invention) with thecarrier which constitutes one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

The compound of the invention will normally be administeredintravenously, orally or by any parenteral route, in the form of apharmaceutical formulation comprising the active ingredient, optionallyin the form of a non-toxic organic, or inorganic, acid, or additionsalt, in a pharmaceutically acceptable dosage form. Depending upon thedisorder and patient to be treated, as well as the route ofadministration, the compositions may be administered at varying doses.

The pharmaceutical compositions must be stable under the conditions ofmanufacture and storage; thus, preferably it should be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.Dependent on the formulation type and administration route chosen, thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g. glycerol, propylene glycol and liquidpolyethylene glycol), vegetable oils, and suitable mixtures thereof.

For example, the compound of the invention may also be administeredorally, buccally or sublingually in the form of tablets, capsules,ovules, elixirs, gels, solutions, emulsions, or suspensions, which maycontain flavouring or colouring agents, for immediate-, delayed- orcontrolled-release applications.

Formulations in accordance with the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

Solutions or suspensions of the compound of the invention suitable fororal administration may also contain excipients e.g. solvents such aswater, ethanol etc., N,N-dimethylacetamide, dispersants e.g. polysorbate80, surfactants, and solubilisers, e.g. polyethylene glycol, Phosal 50PG (which consists of phosphatidylcholine, soya-fatty acids, ethanol,mono/diglycerides, propylene glycol and ascorbyl palmitate). Theformulations according to present invention may also be in the form ofemulsions, wherein a Compound 1 compound may be present in awater-in-oil or oil-in-water emulsion. The oil may be any oil-likesubstance such as e.g. soy bean oil, safflower oil etc., triglyceridessuch as medium chain triglyceride (MCT-oil) such as e.g. coconut oil,palm oil etc or combinations thereof.

Tablets may contain pharmaceutically acceptable excipients such asfillers, binders,dispersing agents, disintegrants, glidants,pH-adjusting agents, stabilisers, taste-masking agents etc. Specificexamples include microcrystalline cellulose, lactose (e.g. lactosemonohydrate or lactose anyhydrous), sodium citrate, calcium carbonate,dibasic calcium phosphate and glycine, butylated hydroxytoluene (E321),crospovidone, hypromellose, disintegrants such as starch (preferablycorn, potato or tapioca starch), sodium starch glycollate,croscarmellose sodium, and certain complex silicates, and granulationbinders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose(HPMC), hydroxy-propylcellulose (HPC), macrogol 8000, sucrose, gelatinand acacia. Additionally, lubricating agents such as magnesium stearate,stearic acid, glyceryl behenate and talc may be included.

A tablet may be made by compression or moulding, optionally with one ormore pharmaceutically acceptable excipients. Compressed tablets may beprepared by compressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), surface-active or dispersing agent. Moulded tablets may bemade by moulding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and may be formulated so as to provideslow or controlled release of the active ingredient therein using, forexample, hydroxypropylmethylcellulose in varying proportions to providedesired release profile.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the compounds of theinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, ethanol, propylene glycol and glycerin, andcombinations thereof.

Formulations suitable for topical administration in the mouth includefilm compositions or lozenges comprising the active ingredient in aflavoured basis, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert basis such as gelatin andglycerin, or sucrose and acacia; and mouth-washes comprising the activeingredient in a suitable liquid carrier.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, emulsions, lotions,powders, solutions, pastes, gels, impregnated dressings, sprays,aerosols or oils, transdermal devices, dusting powders, and the like.These compositions may be prepared via conventional methods containingthe active agent. Thus, they may also comprise compatible conventionalcarriers and additives, such as preservatives, solvents to assist drugpenetration, emollient in creams or ointments and ethanol or oleylalcohol for lotions. Such carriers may be present as from about 1% up toabout 98% of the composition. More usually they will form up to about80% of the composition. As an illustration only, a cream or ointment isprepared by mixing sufficient quantities of hydrophilic material andwater, containing from about 5-10% by weight of the compound, insufficient quantities to produce a cream or ointment having the desiredconsistency.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active agent may be delivered from the patch byiontophoresis.

For applications to external tissues, for example the mouth and skin,the compositions are preferably applied as a topical ointment or cream.When formulated in an ointment, the active agent may be employed witheither a paraffinic or a water-miscible ointment base.

Alternatively, the active agent may be formulated in a cream with anoil-in-water cream base or a water-in-oil base.

For parenteral administration, fluid unit dosage forms or infusions areprepared utilizing the active ingredient and a sterile vehicle, forexample but without limitation water, alcohols, polyols, glycerine andvegetable oils, water being preferred. The active ingredient, dependingon the vehicle and concentration used, can be either colloidal,suspended or dissolved in the vehicle. In preparing solutions the activeingredient can be dissolved in water for injection and sterilised, eg byfilter sterilization, before filling into a suitable vial or ampoule andsealing.

Advantageously, agents such as local anaesthetics, preservatives andbuffering agents can be dissolved in the vehicle. To enhance thestability, the composition can be frozen eg by freeze drying afterfilling into the vial and the water removed under vacuum. The drylyophilized powder is then sealed in the vial and an accompanying vialof water for injection may be supplied to reconstitute the liquid priorto use.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability.

Pharmaceutical compositions of the present invention includeformulations suitable for intraocular administration. These consist of atherapeutically effective quantity of Compound 1, one or morepharmaceutically acceptable excipients or a pharmaceutically acceptablecarrier. Such pharmaceutical compositions may be the conventional dosageform of eye drops or other composition having better bioavailability.Such compositions overcoming the ocular drug delivery barriers andhaving improved ocular bioavailability are e.g. emulsions, ointments,suspensions, aqueous gels, nanomicelles, nanoparticles, liposomes,dendrimers, nanosuspensions, microneedles, and in situ thermosensitivegels.

Parenteral suspensions are prepared in substantially the same manner assolutions, except that the active ingredient is suspended in the vehicleinstead of being dissolved and sterilization cannot be accomplished byfiltration. The active ingredient can be sterilised by exposure toethylene oxide before suspending in the sterile vehicle. Advantageously,a surfactant or wetting agent is included in the composition tofacilitate uniform distribution of the active ingredient.

As seen from the Examples herein, excipients containing carbonate shouldbe avoided, notably in liquid or semi-solid formulations. Preferablycarbonate concentration should be below 0.85 mM.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents. A person skilled in the art will know how tochoose a suitable formulation and how to prepare it (see eg. Remington'sPharmaceutical Sciences 18 Ed. or later). A person skilled in the artwill also know how to choose a suitable administration route and dosage.

The present invention provides a process for preparing a liquidpharmaceutical composition according to any of the preceding claims,said process comprising the steps of:

-   -   a) obtaining methyl        3-[(2-acetylaminoethylthio)carbonyl]propionate (Compound 1) as        the free form or a salt, hydrate, solvate or complex thereof,    -   b) optionally heating to less than 90° C., such as 60° C. or        keeping at room temperature    -   c) adding an aqueous liquid (e.g. phosphate buffer saline at        pH7.4), saline solution or pure water    -   d) optionally aiding dissolution with sonication    -   e) mixing at room temperature

to obtain said pharmaceutical composition.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of a compound of theinvention will be determined by the nature and extent of the conditionbeing treated, the form, route and site of administration, and the ageand condition of the particular subject being treated, and that aphysician will ultimately determine appropriate dosages to be used. Thisdosage may be repeated as often as appropriate. If side effects developthe amount and/or frequency of the dosage can be altered or reduced, inaccordance with normal clinical practice.

All % values mentioned herein are % w/w unless the context requiresotherwise.

Nutricosmetic Compositions Comprising a Compound of the Invention

Nutricosmetics are orally administered products. The present inventionalso provides a nutricosmetic composition comprising the Compound 1compounds. Nutricosmetic compositions comprise Compound 1 in the freeform or a salt, hydrate, solvate or complex thereof together with one ormore orally acceptable diluents or carriers. Nutricosmetic compositionsare very alike pharmaceutical composition for oral administration.

Hence, typical compositions are tablets, capsules, ovules, elixirs,gels, solutions or suspensions.

Cosmeceutical Compositions Comprising a Compound of the Invention

Cosmeceutical compositions are typically administered to the skin ormucosa. Sometimes they may also be given by injections. The presentinvention also provides a cosmeceutical composition comprising theCompound 1 compounds. Cosmeceutical compositions comprise Compound 1 inthe free form or a salt, hydrate, solvate or complex thereof togetherwith one or more orally acceptable diluents or carriers.

Typical cosmeceutical compositions include those mentioned herein abovesuitable for application to the skin, to the mucosa or by injection.

OTHER ASPECTS OF THE INVENTION

The present invention also provides a combination (for example for thetreatment of mitochondrial dysfunction) of a compound of formula (I) ora pharmaceutically acceptable form thereof as hereinbefore defined, andone or more agents independently selected from:

-   -   Quinone derivatives, e.g. Ubiquinone, Idebenone, MitoQ    -   Vitamins e.g. Tocopherols, Tocotrienols and Trolox (Vitamin E),        Ascorbate (C), Thiamine (B1), Riboflavin (B2), Nicotinamide        (B3), Menadione (K3),    -   Antioxidants in addition to vitamins e.g. TPP-compounds (MitoQ),        Sk-compounds, Epicatechin, Catechin, Lipoic acid, Uric acid,        Melatonin    -   Dichloroacetate    -   Methylene blue    -   L-arginine    -   Szeto-Schiller peptides, elamipretide and elamipretide analogs    -   Creatine    -   Benzodiazepines    -   Modulators of PGC-1α    -   Modulators of AMPK    -   Modulators of mitochondrial fission and fusion    -   PPARalfa/beta/gamma-agonists    -   Trolox analogs, carboxamide derivatives    -   Nrf-2 activators    -   NAD⁺ modulators    -   NAD⁺ precursors    -   Ketogenic diet

One other aspect of the invention is that any of the Compound 1compounds as disclosed herein may be administered together with anyother compounds such as e.g. sodium bicarbonate (as a bolus (e.g. 1mEq/kg) followed by a continuous infusion.) as a concomitant medicationto the compounds as disclosed herein.

Lactic Acidosis or Drug-Induced Side-Effects Due to Complex I-RelatedImpairment of Mitochondrial Oxidative Phosphorylation

The present invention also relates to the prevention or treatment oflactic acidosis and of mitochondrial-related drug-induced side effects.In particular the Compound 1 compounds according to the invention areused in the prevention or treatment of a mitochondrial-related drug ortoxin-induced side effects at or up-stream of Complex I, or expressedotherwise, the invention provides according to the invention for theprevention or treatment of drug-induced direct inhibition of Complex Ior of any drug-induced effect that limits the supply of NADH to ComplexI (such as, but not limited to, effects on Krebs cycle, glycolysis,beta-oxidation, pyruvate metabolism and even drugs that effects thetransport or levels of glucose or other complex I related substrates).Mitochondrial toxicity induced by drugs may be a part of the desiredtherapeutic effect (e.g. mitochondrial toxicity induced by cancerdrugs), but in most case mitochondrial toxicity induced by drugs is anunwanted effect. Mitochondrial toxicity can markedly increase glycolysisto compensate for cellular loss of mitochondrial ATP formation byoxidative phosphorylation. This can result in increased lactate plasmalevels, which if excessive results in lactic acidosis, which can belethal. Type A lactic acidosis is primarily associated with tissuehypoxia, whereas type B aerobic lactic acidosis is associated withdrugs, toxin or systemic disorders such as liver diseases, diabetes,cancer and inborn errors of metabolism (e.g. mitochondrial geneticdefects).

Many known drug substances negatively influence mitochondrialrespiration (e.g. antipsychotics, local anaesthetics and anti-diabetics)and, accordingly, there is a need to identify or develop means thateither can be used to circumvent or alleviate the negative mitochondrialeffects induced by the use of such a drug substance. In addition,several chemical agents and gasses negatively influence mitochondrialmetabolism and function.

The present invention provides Compound 1 compounds for use in theprevention or treatment of lactic acidosis and of mitochondrial-relateddrug or toxin-induced side effects. In particular the succinate prodrugsare used in the prevention or treatment of a mitochondrial-relateddrug-induced side effects at or up-stream of Complex I, or expressedotherwise, the invention provides succinate prodrugs for the preventionor treatment of drug-induced direct inhibition of Complex I, otherrespiratory complexes, or of any drug-induced effect that limits thesupply of NADH to Complex I (such as, but not limited to, effects onKrebs cycle, glycolysis, beta-oxidation, pyruvate metabolism and evendrugs that effects the transport or levels of glucose or other Complex Irelated substrates).

As mentioned above, increased lactate plasma levels are often observedin patients treated with drugs that may have mitochondrial-related sideeffects. The present invention is based on experimental results showingthat metformin (first-line treatment for type 2 diabetes and which hasbeen associated with lactic acidosis as a rare side-effect) inhibitsmitochondrial function of human peripheral blood cells at Complex I in atime- and dose-dependent fashion at concentrations relevant formetformin intoxication. Metformin further causes a significant increasein lactate production by intact platelets over time.

Accordingly, the invention provides compounds according to Formula (I)for use in the prevention of treatment of lactic acidosis. However, asthe results reported herein are based on lactic acidosis related todirect inhibition of Complex I or associated with a defect at orup-stream of Complex I, it is contemplated that the compounds accordingto the invention are suitable for use in the prevention or treatment ofa mitochondrial-related drug-induced side-effects at or up-stream ofComplex I. The compounds according to the invention would alsocounteract drug effects disrupting metabolism up-stream of complex I(indirect inhibition of Complex I, which would encompass any drug effectthat limits the supply of NADH to Complex I, e.g. effects on Krebscycle, glycolysis, beta-oxidation, pyruvate metabolism and even drugsthat affect the levels of glucose or other complex I relatedsubstrates). The compounds may also counteract defects down-stream ofComplex I (complex III, IV and V), by increasing proton motive force.

It is contemplated that Compound 1 can be used in industrialapplications, e.g. in vitro to reduce or inhibit formation of lactate orto increase the ATP-availability of commercial or industrial cell lines.Examples include the use in cell culture, in organ preservation, etc.

The compounds according to the invention are used in the treatment orprevention of drug-induced mitochondrial-related side-effects or toincrease or restore cellular levels of energy (ATP) or of succinate, inthe treatment. Especially, they are used in the treatment or preventionof direct or indirect drug-induced Complex I mitochondrial-relatedside-effects. In particular, they are used in the treatment orprevention of lactic acidosis, such as lactic acidosis induced by a drugsubstance.

The invention also relates to a combination of Compound 1 and a drugsubstance that may induce a mitochondrial-related side-effect, inparticular a side-effect that is caused by direct or indirect impairmentof Complex I by the drug substance. Such combination can be used asprophylactic prevention of a mitochondrial-related side-effect or, incase the side-effect appears, in alleviating and/or treating themitochondrial-related side effect.

It is contemplated that Compound 1 will be effective in treatment orprevention of drug-induced side-effects, in particular in side-effectsrelated to direct or indirect inhibition of Complex I.

Drug substances that are known to give rise in Complex I defects,malfunction or impairment and/or are known to have lactic acidosis asside-effect are:

Analgesics including acetaminophen, capsaicin

Antianginals including amiodarone, perhexiline

Antibiotics including linezolid, trovafloxacin, gentamycin

Anticancer drugs including quinones including mitomycin C, adriamycin

Anti-convulsant drugs including valproic acid

Anti-diabetics including metformin, phenformin, butylbiguanide,troglitazone and rosiglitazone,

pioglitazone

Anti-Hepatitis B including fialuridine

Antihistamines

Anti-Parkinson including tolcapone

Anti-psycotics Risperidone,

Anti-schizoprenia zotepine, clozapine

Antiseptics, quaternary ammonium compounds (QAC)

Anti-tuberculosis including isoniazid

Fibrates including clofibrate, ciprofibrate, simvastatin

Hypnotics including Propofol

Immunosupressive disease-modifying antirheumatic drug (DMARD)Leflunomide

Local anaesthetics including bupivacaine, diclofenac, indomethacin, andlidocaine

Muscle relaxant including dantrolene

Neuroleptics including antipsychotic neuroleptics like chlorpromazine,fluphenazine and haloperidol

NRTI (Nucleotide reverse Transcriptase Inhibitors) including efavirenz,tenofovir, emtricitabine, zidovudine, lamivudine, rilpivirine, abacavir,didanosine

NSAIDs including nimesulfide, mefenamic acid, sulindac Barbituric acids.

Other drug substances that are known to have lactic acidosis asside-effects include beta2-agonists, epinephrine, theophylline or otherherbicides. Alcohols and cocaine can also result in lactic acidosis.

Moreover, it is contemplated that the compounds of the invention alsomay be effective in the treatment or prevention of lactic acidosis evenif it is not related to a Complex I defect.

Combination of Drugs and Compounds of the Invention

The present invention also relates to a combination of a drug substanceand a compound of the invention for use in the treatment and/orprevention of a drug-induced side-effect selected from lactic acidosisand side-effect related to a Complex I defect, inhibition ormalfunction, wherein

i) the drug substance is used for treatment of a disease for which thedrug substance is indicated, and

ii) the compound of the invention is used for prevention or alleviationof the side effects induced or inducible by the drug substance, whereinthe side-effects are selected from lactic acidosis and side-effectsrelated to a Complex I defect, inhibition or malfunction.

Any combination of such a drug substance with any compound of theinvention is within the scope of the present invention. Accordingly,based on the disclosure herein a person skilled in the art willunderstand that the gist of the invention is the findings of thevaluable properties of compounds of the invention to avoid or reduce theside-effects described herein. Thus, the potential use of compounds ofthe invention capable of entering cells and deliver succinate andpossibly other active moieties in combination with any drug substancethat has or potentially have the side-effects described herein isevident from the present disclosure.

The invention further relates to

i) a composition comprising a drug substance and a compound of theinvention, wherein the drug substance has a potential drug-inducedside-effect selected from lactic acidosis and side-effects related to aComplex I defect, inhibition or malfunction,

ii) a composition as described above under i), wherein the compound ofthe invention is used for prevention or alleviation of side effectsinduced or inducible by the drug substance, wherein the side-effects areselected from lactic acidosis and side-effects related to a Complex Idefect, inhibition or malfunction.

The composition may be in the form of two separate packages:

A first package containing the drug substance or a compositioncomprising the drug substance and a second package containing theCompound 1 compound of the invention or a composition comprising thecompound of the invention. The composition may also be a singlecomposition comprising both the drug substance and the Compound 1compound of the invention.

In the event that the composition comprises two separate packages, thedrug substance and the Compound 1 compound of the invention may beadministered by different administration routes (e.g. drug substance viaoral administration and compound of the invention by parenteral ormucosal administration) and/or they may be administered essentially atthe same time or the drug substance may be administered before thecompound of the invention or vice versa.

Kits

The invention also provides a kit comprising

i) a first container comprising a drug substance, which has a potentialdrug-induced side-effect selected from lactic acidosis and side-effectsrelated to a Complex I defect, inhibition or malfunction, and

ii) a second container comprising a Compound 1 compound of theinvention, which has the potential for prevention or alleviation of theside effects induced or inducible by the drug sub-stance, wherein theside-effects are selected from lactic acidosis and side-effects relatedto a Complex I defect, inhibition or malfunction.

Method for Treatment/Prevention of Side-Effects

The invention also relates to a method for treating a subject sufferingfrom a drug-induced side-effect selected from lactic acidosis andside-effect related to a Complex I defect, inhibition or malfunction,the method comprises administering an effective amount of a Compound 1compound of the invention to the subject, and to a method for preventingor alleviating a drug-induced side-effect selected from lactic acidosisand side-effect related to a Complex I defect, inhibition or malfunctionin a subject, who is suffering from a disease that is treated with adrug substance, which potentially induce a side-effect selected fromlactic acidosis and side-effect related to a Complex I defect,inhibition or malfunction, the method comprises administering aneffective amount of a Compound 1 compound of the invention to thesubject before, during or after treatment with said drug substance.

Metformin

Metformin is an anti-diabetic drug belonging to the class of biguanides.It's the first line treatment for type 2 diabetes, which accounts foraround 90% of diabetes cases in the USA. The anti-diabetic effect hasbeen attributed to decreasing hepatic glucose production, increasing thebiological effect of insulin through increased glucose uptake inperipheral tissues and decreasing uptake of glucose in the intestine,but the exact mechanisms of action have not been completely elucidated.Despite its advantages over other anti-diabetics it has been related torare cases of lactic acidosis (LA) as side effect). LA is defined as anincreased anion gap, an arterial blood lactate level above 5 mM and a pH≤7.35.

The following list of non-limiting embodiments further illustrate theinvention:

1. Isolated Methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate(Compound 1) being the free form or a salt, hydrate, solvate or complexthereof.

2. The isolated Compound 1 according to embodiment 1, which is a solidproduct.

3. The isolated Compound 1 according to any of the precedingembodiments, which is or comprises a crystalline product such as thepolymorph having the XRPD pattern of Compound 1 Batch 12 (FIG. 7) orhaving the XRPD pattern of Compound 1 Batch 15 (FIG. 8), or having theposition (° 2Theta) being 11.2 (±0.2) and 16.9 (±0.2).

4. The isolated Compound 1 according to any of embodiments 1-2, which isor comprises an amorphous product.

5. The isolated Compound 1 according to any of the precedingembodiments, having a purity of at least 20% w/w at least 30% w/w, atleast 40% w/w, at least 50% w/w, at least 60% w/w, at least 70% w/w, atleast 75% w/w, at least 80% w/w, at least 90% w/w, at least 95% w/w, atleast 97% w/w, at least 98% w/w or at least 99% w/w.

6. The isolated Compound 1 according to any of the precedingembodiments, having a content of related impurities of less than 75%w/w, less than 70% w/w, less than 65% w/w, less than 60% w/w, less than55% w/w, less than 50% w/w, less than 45% w/w, less than 40% w/w, lessthan 35% w/w, less than 30% w/w, less than 25% w/w, less than 20% w/w,less than 15% w/w, less than 10% w/w, less than 5% w/w, less than 3%w/w, less than 2% w/w, or less than 1% w/w.

7. The isolated Compound 1 according to any of the precedingembodiments, having a content of synthesis precursors of less than 50%w/w, less than 40% w/w, less than 30% w/w, less than 25% w/w, less than20% w/w, less than 15% w/w, less than 10% w/w, less than 5% w/w, lessthan 3% w/w, less than 2% w/w, or less than 1% w/w.

8. The isolated Compound 1 according to any of the preceding embodimentshaving a purity sufficient for pharmaceutical use.

9. The isolated Compound 1 according to any of the precedingembodiments, which is the free form.

10. The isolated Compound 1 according to any of embodiments 1-8, whichis a salt.

11. The isolated Compound 1 according to embodiment 10, which is ahydrochloride salt, hydrobromide salt, acetate salt, citrate salt,lactate salt, maleate salt, or malonate salt.

12. The isolated Compound 1 according to any of embodiments 1-8, whichis a hydrate such as a monohydrate.

13. The isolated Compound 1 according to any of the precedingembodiments, for use in humans or animals.

14. The isolated Compound 1 according to any of the precedingembodiments, for use in humans.

15. The isolated Compound 1 according to any of the precedingembodiments, for use in medicine.

16. The isolated Compound 1 according to any of the precedingembodiments, for use as the active pharmaceutical ingredient in apharmaceutical product.

17. The isolated Compound 1 according to any of the precedingembodiments, for use in the treatment or prevention of a metabolicdisease, a disease of mitochondrial dysfunction, a disease related tomitochondrial dysfunction, a mitochondrial disorder, mitochondrialenergy deficiency, drug-induced mitochondrial side effects, cancer,diabetes, traumatic brain injury, cardiac arrest hypoxia, ischemia,stroke, myocardial infarction, acute angina, acute liver injury,coronary occlusion, atrial fibrillation, male infertility and menopausalsymptoms in women.

18. The isolated Compound 1 according to embodiment 17, wherein saiddisease of mitochondrial dysfunction or disease related to mitochondrialdysfunction is selected from

Aging

Alpers Disease (Progressive Infantile Poliodystrophy),

Alzheimer's disease,

Amyotrophic lateral sclerosis (ALS)Autism,

Barth syndrome (Lethal Infantile Cardiomyopathy),

Beta-oxidation Defects, Bioenergetic metabolism deficiency,

Carnitine-Acyl-Carnitine Deficiency,

Carnitine Deficiency,

Creatine Deficiency Syndromes (Cerebral Creatine Deficiency Syndromes(CCDS) including: Guanidinoaceteate Methyltransferase Deficiency (GAMTDeficiency), L-Arginine:Glycine Amidinotransferase Deficiency (AGATDeficiency), and SLC6A8-Related

Creatine Transporter Deficiency (SLC6A8 Deficiency),

Co-Enzyme Q10 Deficiency,

Complex I Deficiency (NADH dehydrogenase (NADH-CoQ reductasedeficiency),

Complex II Deficiency (Succinate dehydrogenase deficiency),

Complex III Deficiency (Ubiquinone-cytochrome c oxidoreductasedeficiency),

Complex IV Deficiency/COX Deficiency (Cytochrome c oxidase deficiency iscaused by a defect in Complex IV of the respiratory chain),

Complex V Deficiency (ATP synthase deficiency),

COX Deficiency, CPEO (Chronic Progressive External OphthalmoplegiaSyndrome),

CPT I Deficiency,

CPT II Deficiency,

Diabetes type II,

Friedreich's ataxia (FRDA or FA),

Glutaric Aciduria Type II,

KSS (Kearns-Sayre Syndrome),

Lactic Acidosis,

LOAD (Long-Chain Acyl-CoA Dehydrogenase Deficiency),

LC-FAOD (Long-Chain Fatty Acid Oxidation Disease)

LCHAD, Leigh Disease or Syndrome (Subacute NecrotizingEncephalomyelopathy),

LHON (Leber's hereditary optic neuropathy),

Luft Disease,

MCAD (Medium-Chain Acyl-CoA Dehydrogenase Deficiency),

MELAS (Mitochondrial Encephalomyopathy Lactic Acidosis and StrokelikeEpisodes),

MERRF (Myoclonic Epilepsy and Ragged-Red Fiber Disease),

METHYLMALONYL-CoA EPIM ERASE DEFICIENCY,

METHYLMALONYL-CoA MUTASE DEFICIENCY,

MITOCHONDRIAL DNA DEPLETION SYNDROME 5,

MITOCHONDRIAL DNA DEPLETION SYNDROME 9,

MITOCHONDRIAL DNA DEPLETION SYNDROME 15 (HEPATOCEREBRAL TYPE)

(1 family),

Maternally inherited diabetes and deafness,

MIRAS (Mitochondrial Recessive Ataxia Syndrome),

Mitochondrial Cytopathy,

Mitochondrial DNA Depletion,

Mitochondrial Encephalopathy including: Encephalomyopathy andEncephalomyelopathy,

Mitochondrial Myopathy,

MNGIE (Myoneurogastointestinal Disorder and Encephalopathy,

NARP (Neuropathy, Ataxia, and Retinitis Pigmentosa),

Neurodegenerative disorders associated with Parkinson's, Alzheimer's orHuntington's disease,

Pearson Syndrome,

Parkinson's disease

Progressive external ophtalmoplegia,

Propionic academia,

Pyruvate Dehydrogenase Deficiency,

POLG Mutations,

Respiratory Chain Deficiencies,

SCAD (Short-Chain Acyl-CoA Dehydrogenase Deficiency),

SCHAD,

VLCAD (Very Long-Chain Acyl-CoA Dehydrogenase Deficiency).

19. The isolated Compound 1 according to embodiment 18, wherein saiddisease of mitochondrial dysfunction or disease related to mitochondrialdysfunction is attributed to complex I dysfunction and selected fromLeigh Syndrome, Leber's hereditary optic neuropathy (LHON), MELAS(mitochondrial encephalomyopathy, lactic acidosis, and stroke-likeepisodes) and MERRF (myoclonic epilepsy with ragged red fibers).

20. The isolated Compound 1 according to any of embodiments 1-17, foruse in the treatment or prevention of metabolic dysfunction.

21. The isolated Compound 1 according to embodiment 20, wherein saidmetabolic dysfunction is diabetes such as defective insulin secretion(Type 2 diabetes).

22. The isolated Compound 1 according to embodiment 20, wherein saidmetabolic dysfunction is drug induced side effects on mitochondria.

23. The isolated Compound 1 according to embodiment 22, wherein saiddrug induced side effects on mitochondria are selected from metformininduced complex I inhibition (lactic acidosis),paracetamol/acetaminophen induced complex I inhibition (liver failure)or drug-induced mitochondrial depletion.

24. The isolated Compound 1 according to embodiment 20, wherein saidmetabolic dysfunction is chemically induced side effects onmitochondria.

25. The isolated Compound 1 according to embodiment 24, wherein saidchemically induced side effects on mitochondria are selected fromrotenone inhibition of complex I (Parkinson like symptoms),pesticide-induced inhibition of respiratory complexes and mitochondrialenzymes, chemical warfare agent-induced inhibition of respiratorycomplexes and mitochondrial enzymes and gaseous poisoning of respiratorycomplexes and mitochondrial enzymes, e.g. carbon monoxide poisoning.

26. The isolated Compound 1 according to embodiment 20, wherein saidmetabolic dysfunction is genetical mitochondrial dysfunction.

27. The isolated Compound 1 according to embodiment 26, wherein saidgenetical mitochondrial dysfunction is selected from dysfunctionalenergy production due to decreased number of mitochondria, dysfunctionalmitochondrial transcription factors, dysfunctional transcription factorsfor nuclear DNA encoded mitochondrial proteins, mitochondrial membraneproteins that contribute to the stabilization of large mitochondrial DNA(mtDNA)-protein complexes called nucleoids, dysfunctional energyproduction, pyruvate dehydrogenase deficiencies, deficiency of complexI, II, III or IV, or an enzyme deficiency like e.g. pyruvatedehydrogenase deficiency, dysfunction of enzymes involved in succinatesynthesis, e.g. propionyl CoA carboxylase, methylmalonyl CoA mutase andsuccinyl CoA synthetase.

28. The isolated Compound 1 according to embodiment 27, where saideffective amount is in the range from from 1 mg to 5.0 g per day, from10 mg to 2.0 g per day, from 25 mg to 1 g per day, from 50 mg to 500 mgper day, from 100 mg to 1000 mg per day, from 250 mg to 1000 mg per day,or from 50 mg to 500 mg per day of Compound 1 or a salt, hydrate,solvate or complex thereof.

29. The isolated Compound 1 according to any of embodiments 15-28,wherein said Compound 1 or a salt, hydrate, solvate or complex thereofis administered to said subject from one time per day to 10 times perday, or from one time per day to 4 times per day.

30. The isolated Compound 1 according to any of embodiments 15-29,wherein said treatment or prevention is pre-treatment, e.g. the usebefore surgery, the use before planned medical intervention with a highmetabolic demand, and before subject entering a war zone or otherhazardous environment.

31. The isolated Compound 1 according to any of embodiments 15-30,wherein said treatment or prevention is a chronic treatment.

32. The isolated Compound 1 according to any of embodiments 1-14, fornon-pharmaceutical use in humans or animals.

33. The isolated Compound 1 according to embodiment 19, for use as acosmeceutical or nutricosmetics.

34. The isolated Compound 1 according to any of embodiments 28-29, foruse as an energy drink or a cream.

35. Composition comprising the isolated Compound 1 according to any ofthe preceding embodiments.

36. Cosmeceutical comprising the isolated Compound 1 according to any ofembodiments 1-14.

37. Nutricosmetics comprising the isolated Compound 1 according to anyof embodiments 1-14.

38. Energy drink comprising the isolated Compound 1 according to any ofembodiments 1-14.

39. Pharmaceutical composition comprising isolated Compound 1 accordingto any of embodiments 1-33.

40. A process for preparing isolated Compound 1 according to any ofembodiments 1-33, said process comprising the steps of:

a) reacting N-acetyl cysteamine and monomethyl succinate, in thepresence of a coupling reagent, in organic solvent, between 0° C. and100° C.

b) isolating Compound 1, so as to provide isolated Compound 1.

41. The process according to embodiment 40, wherein step a) is conductedwhere independently the solvent is dichloromethane, the coupling agentis carbonyldiimidazole and the temperature is 15-30° C.

42. The process according to any of embodiments 40-41 wherein step b)comprises extraction with an aqueous acidic solution (optionally 20%ammonium chloride) and then extracting the organic layer with anotheraqueous medium (suitable brine or water).

43. The process according to embodiment 42 wherein the organic layer isremoved in vacuo and the residue dissolved in an organic solvent withsuitable dissolution properties for crystallization, such asmethyl-tert-butylether (MTBE).

44. The process according to embodiment 42 wherein the solution iscooled, suitably to approximately 5° C. and an antisolvent is added,such as n-heptane, after stirring for a period of time, suitablyapproximately 24 hours, compound 1 is harvested by filtration and washedwith an antisolvent.

45. The isolated Compound 1 according to any of embodiments 1-3 wherethe position (° 2Theta) is 11.2 (±0.2) and 16.9 (±0.2).

46. A pharmaceutical composition according to embodiment 39, which is asolid formulation.

47. The pharmaceutical composition according to embodiment 46, which isa solid formulation for reconstitution prior to use.

48. The pharmaceutical composition according to embodiment 46, which isan aqueous formulation.

49. The pharmaceutical composition according to embodiment 48, which isan aqueous Phosphate Buffered Saline (PBS) formulation.

50. The pharmaceutical composition according to any of embodiments46-49, which has a concentration of Compound 1 of at least 10% w/w, atleast 30% w/w, at least 50% w/w, at least 60% or at least 70% w/w.

51. The pharmaceutical composition according to any of embodiments46-50, which is for oral administration, for subcutaneousadministration, for intravenous administration, for parenteraladministration, for ocular administration or for topical administration.

52. The pharmaceutical composition according to embodiment 51, which isa drink or a gel.

53. The pharmaceutical composition according to any of embodiments46-52, which comprises from 1 mg to 5.0 g, from 10 mg to 2.0 g, from 25mg to 1 g, from 50 mg to 500 mg, from 100 mg to 1000 mg, from 250 mg to1000 mg, or from 50 mg to 500 mg of Compound 1 or a salt, hydrate,solvate or complex thereof.

54. The pharmaceutical composition according to any of embodiments46-53, which is an immediate release formulation.

EXAMPLES

General methods, materials and assays.

HPLC method for purity analysis.

HPLC Method 1

Solvent A is Water+0.1% NH₄OH

Solvent B is 2.5 L Acetonitrile+130 ml H₂O+0.1% NH₄OH

Gradient: T=0 minutes, B %=5, flow rate=1 ml/min; T=0.1 minutes, B %=5,flow rate=1 ml/min; T=9.5 minutes, B %=95, flow rate=1 ml/min; T=10.2minutes, B %=95, flow rate=1 ml/min; T=10.3 minutes, B %=95, flowrate=1.5 ml/min; T=11.1 minutes, B %=95, flow rate=1.5 ml/min; T=11.15minutes, B %=5, flow rate=1.5 ml/min; T=11.5 minutes, B %=5, flowrate=1.5 ml/min;

Column is a Waters XSelect CSH C18 3.5 um, 2.1 mm×50 mm.

Absorbance is monitored at 234 nm on a diode array detector.

1 mg/ml sample conc, 1γl injection volume

HPLC Method 2

Solvent A is Water+1.57 g NH₄HCO₂+5 ml Formic acid

Solvent B is 2.5 L Acetonitrile+130 ml H₂O+4.5 ml Formic acid

Gradient: T=0 minutes, B %=0, flow rate=1 ml/min; T=1 minutes, B %=0,flow rate=1 ml/min; T=9.5 minutes, B %=20, flow rate=1 ml/min; T=10.3minutes, B %=95, flow rate=1 ml/min; T=10.5 minutes, B %=95, flowrate=1.5 ml/min; T=11.0 minutes, B %=95, flow rate=1.5 ml/min; T=11.05minutes, B %=0, flow rate=1.5 ml/min; T=11.5 minutes, B %=0, flowrate=1.5 ml/min;

Column is a Waters XSelect CSH C18 3.5 um, 2.1 mm×50 mm.

Absorbance is monitored at 230 nm on a diode array detector.

1 mg/ml sample conc, 1γl injection volume

Example 1—Synthesis of methyl3-[(2-acetylaminoethylthio)carbonyl]propionate (Compound 1) DetailedDescription of the Synthesis and Isolation of Compound 1

Compound 1 has been made by three separate methods (A, B and C below).

Method A

To a solution of 2-aminoethanethiol hydrochloride (226 g, 2 mol), KOH(114 g, 2 mol) and NaHCO₃ (168 g, 2 mol) in water (4 L) was added aceticanhydride (204 g, 2 mol) dropwise. The mixture was stirred at roomtemperature for 45 minutes. The reaction mixture was extracted withEtOAc (8×2 L), dried over MgSO₄ and the solvent was removed to giveintermediate 1 (190 g, 80% yield) as slight yellow oil under reducedpressure.

To a solution of 4-methoxy-4-oxobutanoic acid (209 g, 1.583 mol) andHOBT (214 g, 1.583 mol) in dichloromethane (4 L) was addedN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (304 g,1.583 mol). The mixture was stirred at room temperature for 2 hours.Intermediate 1 (189 g, 1.583 mol) was added dropwise. The mixture wasstirred at room temperature for 2 hours. triethylamine (160 g, 1.583mol) was added dropwise. The mixture was stirred at room temperatureovernight. The resulting mixture was washed with water (2 L) andsaturated solution of NaHCO₃ (2×2 L), dried over Na₂SO₄ and concentratedunder reduced pressure to afford crude compound 1 (350 g) as yellow oil.The crude compound 1 was purified by silica gel column chromatography(2000 g silica gel, eluting with CH₂Cl₂/MeOH=100/1 to 80/1) to givecompound 1 (201 g, 94.9% in LCMS) as white solid. Crude side cuts fromthe purification (110 g) were purified by silica gel columnchromatography (1200 g silica gel, eluting with CH₂Cl₂/MeOH=100/1 to80/1) to give compound 1 (40 g, 96.7% in LCMS) as white solid.

Method B

Acetic anhydride was added dropwise (7.14 g, 0.07 mol) to a solution of2-aminoethanethiol hydrochloride (11.3 g, 0.1 mol), KOH (5.6 g, 0.1 mol)and NaHCO₃ (5.88 g, 0.07 mol) in water (200 mL) at room temperature. Themixture was stirred at room temperature for 45 minutes. The reactionmixture was extracted with EtOAc (8×200 mL), dried over MgSO₄ (1 h) andthen the solvent was removed in vacuo at 50° C. to give crudeintermediate 1 (7 g, 84% yield) as slight yellow liquid.

1,1′-Carbonyldiimidazole (11.34 g, 0.07 mol) was added portion wise to asolution of 4-methoxy-4-oxobutanoic acid (9.24 g, 0.07 mol) indichloromethane (200 mL). The mixture was stirred at room temperaturefor 1 hour. intermediate 1 (7 g, 0.059 mol) was added dropwise and thenthe mixture was stirred at room temperature for 3 hours. The resultingmixture was washed with HCl (1N, 3×150 mL) and saturated solution ofNaHCO₃ (3×150 mL), dried over Na₂SO₄ (1 h) and then the solvent wasremoved in vacuo at 50° C. to afford 9.5 g of compound 1 as yellowsolid.

Method C

To the solution of KOH (0.71 kg, 13.2 mol) and Na₂CO₃ (1.00 kg, 9.43mol) in water (15 L) 2-aminoethanethiol hydrochloride (1.5 kg, 13.2 mol)was added. To the resulting clear dark violet solution acetic anhydride(0.96 kg, 9.43 mol) was added dropwise at +22° C. keeping the internaltemperature below +30° C. during addition (addition time was 24minutes). The reaction mixture was stirred at +20° C. for 1 h 35 min.Dichloromethane (23 L) was added and the mixture was stirred at +28±2°C. for 20 minutes. The layers were separated. Aqueous phase wasextracted with dichloromethane (2×15 L) adjusting the internaltemperature to +28±2° C. during extractions. The organic phases werecombined, and solvent was removed in vacuo. Afterwards intermediate 1was dried under vacuum at +40° C. for 18 hours. Yield 996 g andpurity >97 area-% (GC). Crude intermediate 1 was obtained as brown oil.

Distillation: 933 g of intermediate 1 was distilled using thin layerdistillation unit under the following conditions: T=+110° C., P=1 mbar,rate 202 g/h. Intermediate 1 was obtained as clear colourless oil.

To the solution of 4-methoxy-4-oxobutanoic acid (1.33 kg, 10.07 mol) inDCM (10 L) 1,1′-carbonyldiimidazole (CDI) (1.63 kg, 10.07 mol) was addedportionwise. Intensive foaming and gas evolution were observed duringaddition. After the addition was finished the mixture was stirred at +20to +25° C. for 1 hour. Intermediate 1 (1.00 kg, 8.39 mol) solution indichloromethane (5 L) was added keeping the internal temperature below+30° C. The reaction mixture was stirred at +20 to +25° C. for 2 hours.20% NH₄Cl aqueous solution (10 L) was added and the mixture was stirredfor 20 minutes. The layers were separated. Organic phase was extractedwith 13% NaCl aqueous solution and water, subsequently (10 L and 5 L,individually). Afterwards the solvent (DCM) was changed to MTBE bydistillation. Solution of compound 1 in MTBE (approximately 6 L) wasgradually cooled to +5° C. Crystallization started when the internaltemperature reached +12° C. To the slurry n-heptane (15 L) was added andthe mixture was stirred at 0 to +5° C. for 20 hours (overnight). Slurrywas filtered and the filter cake was washed with N-heptane (2×3 L).Product was dried by pulling air through it for 42 hours. Yield was 1.26kg (64%) and purity 98.5 area-% (HPLC).

Several batches of Compound 1 were prepared by the above describedsynthesis. The batches were purified by different purification methods,as set out in Methods A, B and C.

Batch 3 (or compound 1-s3) was prepared via Method A.

Batch 12 (or compound 1-s12), 13 (or compound 1-s13) and 14 (or compound1-s14) were prepared by method B.

Batch 15 (or compound 1-s15), 16, and 17 were prepared by method C.

Example 2—Characterization of Compound 1 from Different Batches

Method of Crystallinity by Batch number generation XRPD data XRPD  2Method A  3 Method A Figure 19 74.4%  4 Method A  5 Method B  6 Method B11 Method A then puri- fied by Method B 12 Method B Figure 11 13 MethodB Figure 21 70.2% 14 Method B Figure 22 56.2% 15 Method C Figure 12 16Method C Figure 24 65.2% 17 Method C then puri- Figure 25 63.7% fied byprep-HPLC and lyophilised 18 (stored batch Batch 12, stored as Figure 2070.4% 12) solid at −20° C. for 20 months 19 (stored batch Batch 15,stored as Figure 23 69.7% 15) solid at −20°C. for 14 months

It should be mentioned that the temperature increases during XRPDanalysis. As Compound 1 has a low melting point (and the amorphous formis contemplated to have a lower melting point than the crystallineform), the degree of crystallinity given in the table above may beregarded as minimum values.

Batch 3.

In TGA analysis the Batch 3 had a loss of 0.04% by weight attemperatures 20-150° C. FIGS. 1-3 shows the spectra from LCMS analysisof Batch 3. The melting point of Batch 3 was 50.4° C. as determined fromDifferential Scanning calorimetry (DSC).

Batch 12.

Batch 12 was analysed by same methods as used for analysis of the abovedescribed Batch 3.

The loss in TGA analysis was 0.12% by weight at temperatures 20-150° C.Results from LCMS are depicted in FIGS. 4-6, the purity by qNMR was96.1% and the melting point was 48.6° C.

Batch 13.

Batch 13 was analysed by same methods as used for analysis of the abovebatches. The loss in TGA analysis was 0.18% by weight at temperatures20-150° C. Spectra from LCMS are not shown but results summarised inTable 5. The purity by qNMR was 96.3% and the melting point was 49.0° C.

Batch 14

Batch 14 was analysed by same methods as used for analysis of the abovebatches. The loss in TGA analysis was 0.45% by weight at temperatures20-150° C. Spectra from LCMS are not shown but results summarised inTable 5. The purity by qNMR was 91.6% and the melting point was 46.9° C.

Batch 15

Batch 15 was analysed by some of the same methods as used for analysisof the above batches.

Spectra from LCMS are not shown but results summarised in Table 5. Thepurity by qNMR was 98.9% and the melting point was 39° C.

Comparison of Batch Properties: Batches 3, 12, 13, 14, 15 and 16.

Table 5 also summarizes the purities, melting point and visualdescription of the solid Compound 1. The solid Compound 1 batchesappeared as white, free-flowing powder, c.f. Table 5.

Additionally, Table 5 shows the solubility of the prepared Compound 1batches were all at least 366-398 mg/ml and the appearance of such aformulation in water to visually appear as a clear and transparent ortranslucent solution.

TABLE 5 Summary of results from analyses of different batches ofCompound 1. Batch 3 Batch 12 Batch 13 Batch 14 Batch 15 Batch 16 FinalPurified Washed, Washed, Washed, Washed, Washed, Purifi- by silica driedand dried and dried and precipitated precipitated cation gel columnsolvent solvent solvent and and Method chroma- removal removal removaldried. dried. tography Purity by 99.1 97.7 96.7 99.3 98.5 98.1 high pHHPLC (%) Purity 98.1 96.1 96.3 91.7 98.9 95.6 assay by qNMR (%) Melting50.4 48.6 49.0 46.9 39 Point (° C.) Visual White, Large Large LargeWhite, White, Description free- white white white free- free- of Formflowing waxy waxy waxy flowing flowing powder; lumps; lumps; lumps;powder powder some some some some ‘clumped’ white, white, white,material free- free- free- flowing flowing flowing powder powder powder400 mg 381 378 395 366 414 435 Com- pound in 0.7 ml Water, MeasuredConc. (mg/ml) Visual Colour- Cloudy, Colour- Colour- Colour- Colour-Descrip- less, trans- less, less, less, less, tion of trans- lucenttrans- trans- trans- trans- Formula- parent solution parent parentparent parent tion in solution solution solution solution solution Water

TABLE 6 Summary of the analysis of the Compound 1 batches re purity andimpurities (LCMS2 High pH Impurity Profiling). RT (min)  0.69  1.57 1.86  2.18  2.22  6.24 RRT (min)  0.439  1.000  1.185  1.389  1.414 3.975 Apparent molecular 161.0  233.0  319.9  247.0  305.0  278.0 weight Structure (proposed from m/z)

TBC TBC TBC TBC % at 234 nm, batch 3  0.52 99.14  0.35 % at 234 nm,batch 12 97.65  1.41  0.24  0.38  0.32 % at 234 nm, batch 13  0.47 96.71 2.44  0.39 % at 234 nm, batch 14  0.34 99.34  0.32

Example 3—Preparation of Aqueous Formulations of Compound 1

Formulation Protocol

-   -   1. Weigh out the required amount of Compound 1 then add the        required amount of excipient (0.9% w/v saline, 100 mM PBS pH        7.4, water) to the solid Compound 1 to give the required mg/ml        concentration of Compound 1. For example, for a 400 mg/ml        formulation of Compound 1 in water, weigh out 400 mg of Compound        1 and add 0.7 ml of water.    -   2. Sonicate the solution for 10 mins and then shake for 20 mins        to ensure that Compound 1 is fully dissolved.    -   3. The solution can be centrifuged (13000 rpm, 10 mins) to        remove any particulates if required.    -   4. The solution can be sterile filtered if required.

TABLE 7 Data for formulation in water of different batches of Compound 1prepared according to the protocol above Target Conc. Inj. Vol. DilutionCalculated Purity Sample (mg/ml) (μl) factor Conc. (mg/ml) (%) Batch 3in tap 400 2 1000 381 98.8 water Batch 12 in 400 2 1000 378 99.2 tapwater Batch 13 in 400 2 1000 395 99.1 tap water Batch 14 in 400 2 1000366 99.4 tap water

Formulation 50% w/v

Four batches of Compound 1 were formulated in PBS at 50% w/v.

˜500 mg of compound was weighed into a vial, ˜500 μl 100 mM PBS pH 7.4added and the vial was sonicated for 10 mins then shaken for 20 mins. Asample was then removed, diluted 1/2000 and the concentration calculatedby HPLC analysis.

TABLE 8 Preparation of Compound 1 formulations in PBS at pH 7.4. Theconcentration of Compound 1 was measured in the soluble formulation byHPLC. Amount Amount 100 mM Calculated Compound 1 PBS pH concentrationSample added (mg) 7.4 added (μl) by HPLC, (mg/ml) Batch 3 525 525 535Batch 4 514 514 526 Batch 5 494 494 531 Batch 6 481 481 543

Formulation Liquid Compound 1

Compound 1 batch 3 was heated in an oven to 60° C. for 20 mins, at whichpoint it became a translucent pale-yellow liquid. 100 mM PBS pH 7.4 (20%v/v) was added and the solution was mixed for 20 mins on a shaker. Afterthis time, the solution had cooled to room temperature and remained atranslucent liquid. The solution was placed at 4° C. for 72 hours.Observation after this time confirmed that it remained a translucentliquid.

Formulation Summary

The amount of Compound 1 that can be formulated in aqueous solutionssuch as 0.9% w/v saline, 100 mM PBS pH 7.4 or water seems to have noreachable limit. This can possibly be explained by the melting point ofCompound 1 which was measured to approx. 47-50° C. in several batches.When the aqueous solution is added to the solid, it disrupts theintramolecular interactions of Compound 1 molecules it becomes misciblewith water.

Example 4—Gel Formulation

Formulate Compound 1 into gel packs at 2.25 mg/ml.

Experimental Details

One HydroGel gel pack (Clear H₂O hydrogel, 8 oz pouch, HydroGel,Portland, Me.) was taken and divided up into falcon tubes for differentexperiments.

Firstly, blue food dye was used to see how easily an aqueous solutioncould be mixed into the gel. Two samples of the gel were taken, one waskept at RT, the other was melted in the microwave (1 min). The blue fooddye (1% v/v) was added and the solutions were mixed. Mixing is much moreefficient when the gel has been melted. The dye can be fullyincorporated after mixing for <10 s.

Next, the dye solution was replaced for a solution of Compound 1.

Water was added to Compound 1 (225 mg/ml, 100× required concentration)and sonicated for 20 mins then shaken for 30 mins. A sample was takenfor HPLC analysis to check the concentration.

TABLE 9 Compound 1 measured concentration in water stock. Expected conc.Inj. Vol. Dilution Sample (mg/ml) (ul) factor Calc. mg/ml Purity (%)Water stock 225 1 100 222.749 98.8 for addition into gel

The analysis showed that Compound 1 had been fully solubilised.

Another 2 portions of the gel were melted, and the Compound 1 watersolution was added (1% v/v). The gels were shaken for the same length oftime as when the dye was added (10 s). The gels were left to set.

Once the gels had set, samples were taken for HPLC analysis to checkCompound 1 was evenly distributed.

Sampling Procedure

-   -   1. A sample of the gel (100 mg) was added to an Eppendorf and        MeOH was added (0.9 ml, 1/10 dilution)    -   2. The sample was shake on a vibrax for 30 mins    -   3. The sample was centrifuged (13000 rpm, 10 mins)    -   4. The supernatant was taken for HPLC analysis

TABLE 10 Compound 1 measured concentration in gel samples. ExpectedDilution Calc. Sample conc. (mg/ml) Inj. Vol. (ul) factor mg/ml Purity(%)  4° C. 2.25 5 10 2.07 99.4 37° C. 2.25 5 10 2.03 99.5

The concentration of Compound 1 was slightly lower than the 2.25 mg/mlexpected, but the two samples are in good agreement, which suggestsCompound 1 has been evenly distributed and that the dilution factor isslightly out.

One of the gel samples was kept at RT (room temperature) and the otherat 4° C. to test the stability of Compound 1 in the gel.

Stability of Gel Formulation

The gel was sampled as above at regular timepoint to check the stabilityat both 4° C. and RT. Data from these experiments are shown in Tables11-12.

TABLE 11 Compound 1 concentrations in gel samples taken over 20 daysstorage at 4° C. Expected Inj. AUC Purity at Time conc. Vol. DilutionCalc. (% of 230 nm (days) (mg/ml) (ul) factor mg/ml T = 0) (%) 0 2.25 510 2.071 100.0 99.4 1 2.25 5 10 2.024 97.8 99.4 5 2.25 5 10 1.937 93.599.4 7 2.25 5 10 1.979 95.6 99.4 11 2.25 5 10 2.007 96.9 99.4 14 2.25 510 2.167 104.7 99.4 20 2.25 5 10 2.218 107.1 99.5

TABLE 12 Compound 1 concentrations in gel samples taken over 20 daysstorage at RT (approx. 20° C.). Expected Inj. AUC Purity at Time conc.Vol. Dilution Calc. (% of 230 nm (days) (mg/ml) (ul) factor mg/ml T = 0)(%) 0 2.25 5 10 2.034 100.0 99.5 1 2.25 5 10 1.746 85.8 99.5 5 2.25 5 102.053 100.9 99.4 7 2.25 5 10 1.721 84.6 99.2 11 2.25 5 10 1.889 92.999.1 14 2.25 5 10 2.739 134.6 99.4 20 2.25 5 10 2.273 111.7 99.4

The general trend in the data, especially the purity at 230 nm datasuggests Compound 1 is stable in the gel formulation for at least 20days. The AUC data has more error due to inaccuracies in weighing gelsamples for extraction and possibly differences in localisedconcentration of Compound 1 in the gel.

Protocol for Preparation of Gel Formulation

Prepare a 100× concentrated solution of Compound 1 in water, so it canbe added to the gel at 1/100 of the volume of the gel. The example givenis for a 2.25 mg/ml final concentration in a 200 ml gel pack, thereforerequires 2 ml of 100× Compound 1 in water (225 mg/ml). It is alsosuggested that a food dye is added to the stock solution and thecombined solution is injected into the gel pack (provided it will causeno adverse effect to the study). This gives a visual check that theCompound 1 solution has been evenly distributed in the gel. If the fooddye is included, it is recommended that the food dye is also added tothe gel of control group.

-   -   1. Weigh out 500 mg Compound 1 into a 3 ml vial (or similar)    -   2. Add 2 ml water (the addition of 2 ml water to 500 mg Compound        1 accounts for the volume of the solid Compound 1 and has been        confirmed by HPLC calibration curve to be 225 mg/ml), sonicate        for 20 mins, then shake for 30 mins (the solution may remain        slightly cloudy). Add 1 ml of a natural food dye (the food dye        will help to show that even distribution has been achieved).    -   3. This solution can be sterile filtered if necessary    -   4. Heat the unopened gel pack by submersing in 70° C. water for        10 mins and the gel will become a mobile liquid    -   5. Take up the Compound 1 solution into a needle/syringe        (with/without the food dye)    -   6. Inject the Compound 1 solution (and the food dye) into the        gel pack by piercing a small hole in the gel pack with the        needle    -   7. Re-seal the gel pack at the injection site with tape    -   8. Shake the gel pack vigorously for 5 mins to get even        distribution of Compound 1 (if the food dye is included it will        become evident when the injected solution is evenly distributed        by a colour change of the gel)    -   9. Allow the gel to solidify, this takes around 45 mins at room        temperature    -   10. Use the gel or store sealed at 4° C. (it is recommended that        the gel is stored at 4° C. for a maximum of 14 days once opened)    -   11. Compound 1 is stable in the gel for at least 14 days at        4° C. and room temperature

Test of Protocol

The gel formulation was prepared according to the protocol and HPLCanalysis showed that the correct concentration of Compound 1 wasachieved in the water stock and the gel formulation.

TABLE 13 Compound 1 concentrations the water stock solution and the gelformulation. Expected conc. Inj. Vol. Dilution Calc. Purity at Sample(mg/ml) (ul) factor mg/ml 230 nm (%) Water stock 225 2 200 229.496 99.0for addition into gel Gel formu- 2.25 5 10 2.355 99.7 lation

2 ml of the water stock was added to 1 ml of food dye. This was dilutedand analysed by HPLC. The AUC for the water stock diluted with food dyewas 0.63 times that of the water stock before the dilution, indicatingthat Compound 1 remains soluble when diluted with food dye.

Outcome/Conclusion

Compound 1 can be formulated in aqueous gel packs at 2.25 mg/ml and isstable at both 4° C. and RT for at least 20 days.

Example 5—Saline Formulation of Compound 1

-   -   1. Weigh out 400 mg Compound 1 and add 0.7 ml saline (0.9% w/v)    -   2. Sonicate for 20 mins then shake for 30 mins or until all        compound is soluble by eye

Compound 1 Freeze/Thaw Stability

Method:

-   -   1. Saline (0.9% w/v) was added to Compound 1 (1 mg/ml) then        sonicated for 10 mins until fully dissolved    -   2. A sample was taken for HPLC analysis (F/T 0)    -   3. The solution was frozen at −80° C. overnight.    -   4. The solution was thawed, and a sample was taken for HPLC        analysis (F/T 1)    -   5. Steps 3&4 were repeated for 3 cycles

TABLE 14 Purity of Compound 1 upon Freeze/Thaw (FT) cycles. ExpectedInj. FT conc. Vol. Dilution Calc. Purity Sample cycle (mg/ml) (ul)factor mg/ml (%) Compound 0 1 2 1 1.157 98.9 1 in 1 1 2 1 1.148 98.8saline 2 1 2 1 1.153 98.9 3 1 2 1 1.146 98.8

The results (Table 14) show that there is no significant change in assayor purity of Compound 1 in saline, so Compound 1 is stable for at least3 freeze/thaw cycles.

Compound 1 RT Stability

Method:

-   -   1. Saline (0.9% w/v) was added to Compound 1 (1 mg/ml) then        sonicated for 10 mins until fully dissolved    -   2. A sample was taken for HPLC analysis (T=0)    -   3. The solution was stored at RT    -   4. Samples were taken periodically for HPLC analysis of        stability

Results:

The results showed that there is no significant change in assay orpurity of Compound 1 in saline (data not depicted), so Compound 1 isstable for at least 14 days at room temperature.

Compound 1 200-500 mg/ml Formulation

Method:

-   -   1. Saline (0.9% w/v) was added to Compound 1 batch 11 in        different amounts then sonicated for 10 mins—the samples        remained slightly cloudy    -   2. The samples were shaken for 30 mins at which point the        solutions became translucent    -   3. The solutions were diluted for HPLC analysis

Results:

TABLE 15 Purity of Compound 1 at increasing concentrations in salinesolution. RT lnj. Vol. Dilution Calc. Purity Sample (min) (ul) factormg/ml (%) 400 mg Compound 1 in 1 ml 8.21 5 1000 261 98.7 saline 400 mgCompound 1 in 0.7 ml 8.21 5 1000 403 98.7 saline 400 mg Compound 1 in0.4 ml 8.21 5 1000 545 98.6 saline

The results show (Table 15) that over 500 mg/ml Compound 1 in salinesolution can be reached.

Protocol for Transfer (400 mg/ml Formulation)

-   -   3. Weigh out 400 mg Compound 1 batch 11 and add 0.7 ml saline        (0.9% w/v)    -   4. Sonicate for 20 mins then shake for 30 mins or until all        compound is soluble by eye

Outcome/Conclusion

Compound 1 Freeze/Thaw Stability

Compound 1 is stable for at least 3 freeze/thaw cycles.

Compound 1 RT Stability

Compound 1 is stable for at least 14 days at room temperature (datacollection to be continued).

Compound 1 400 mq/ml Formulation

Over 500 mg/ml Compound 1 in saline can be reached.

Example 6—Compound 1 Stability in Water and DMSO

Compound 1 was separately dissolved in water and DMSO at a concentrationof 1 mg/ml and stored at RT, 37° C. and 65° C. for stability over a 40day period.

TABLE 16 Compound 1 in water at RT in the dark (conc. 1 mg/ml) withpurity measurements. Time AUC at 230 AUC (% of Purity at 230 nm Purityby LCMS (days) nm T = 0) (%) (%) 0 9041 100.0 100.0 100.0 1 8847 97.9100.0 99.2 2 8807 97.4 100.0 98.3 4 8045 89.0 100.0 99.0 7 8588 95.0100.0 99.2 11 8554 94.6 100.0 98.7 14 8662 95.8 100.0 95.6 17 8873 98.1100.0 98.1 23 8561 94.7 100.0 98.9 38 8555 94.6 100.0 98.7

Small loss in purity and assay is noted in Compound 1 in water at roomtemperature over the period of 38 days.

TABLE 17 Compound 1 in water at 37° C. in the dark (conc. 1 mg/ml) withpurity measurements. Time AUC at 230 AUC (% of Purity at 230 nm Purityby LCMS (days) nm T = 0) (%) (%) 0 9044 100.0 100.0 100.0 1 9798 108.3100.0 99.6 2 8681 96.0 100.0 97.5 4 8156 90.2 100.0 99.0 7 9106 100.7100.0 98.9 11 8577 94.8 100.0 98.4 14 7777 86.0 100.0 96.9 17 7928 87.7100.0 93.2 23 7567 83.7 100.0 98.4 38 7579 83.8 100.0 98.3

Loss in purity and assay is noted in Compound 1 in water at 37° C. overthe period of 38 days. Loss in assay is more significant than thatobserved in water at room temperature.

TABLE 18 Compound 1 in water at 65° C. in the dark (conc. 1 mg/ml) withpurity measurements. Time AUC at 230 AUC (% of Purity at 230 nm Purityby LCMS (days) nm T = 0) (%) (%) 0 8741 100.0 100.0 100.0 1 8614 98.5100.0 98.3 2 8410 96.2 100.0 97.3 5 7512 85.9 100.0 95.9 8 8750 100.1100.0 99.1 14 9095 104.0 100.0 99.0 29 4521 50.0 100.0 89.1

Loss in purity and assay is noted in Compound 1 in water over the periodof 29 days. Loss in purity and assay is more significant than thatobserved in water at 37° C.

TABLE 19 Compound 1 in DMSO at 37° C. in the dark (conc. 1 mg/ml) withpurity measurements. AUC Purity at Purity Time AUC at (% of 230 nm byLCMS (days) 230 nm T = 0) (%) (%) 0 16363 100.0 100.0 98.4 1 17465 106.7100.0 98.4 4 17041 104.1 100.0 98.2 8 17644 107.8 100.0 98.2 11 17434106.5 100.0 99.0 14 17557 107.3 100.0 98.2 22 17353 106.1 100.0 98.0 3717904 109.4 100.0 97.6

No significant loss in purity or assay is noted in Compound 1 in DMSO at37° C. over the period of 37 days.

TABLE 20 Compound 1 in DMSO at 65° C. in the dark (conc 1 mg/ml) withpurity measurements. AUC Purity at Purity Time AUC at (% of 230 nm byLCMS (days) 230 nm T = 0) (%) (%) 0 15834 100.0 100.0 98.1 1 16974 107.2100.0 98.3 2 16185 102.2 100.0 96.8 5 17175 108.5 100.0 99.2 8 1441591.0 100.0 96.7 14 13846 87.4 100.0 93.9 29 16472 100.7 100.0 97.7

No significant loss in purity or assay is noted in Compound 1 in DMSO at65° C. over the period of 29 days.

Example 7—Analysis of Compound 1

Material from Method C (Batch 15) from Example 1 was analysed by XRPD.The data are shown in FIG. 12 and shows crystalline material.

Material from Method A (Batch 12) was also analysed by XRPD and appearedto show crystalline material with the same polymorph (FIG. 11).

Example 8—Comparison of Solubility of Compound 1 with Other SuccinateProdrugs

Solubility of Compound 1 in aqueous formulations in comparison to othersuccinate prodrugs was assessed by dissolving solid material in aqueousformulations and measuring the quantity in solution by HPLC (−MS).Compound 1 was seen to dissolve in water at over 350 mg/mL, PBS (pH 7.4)at 190 mg/mL and over 500 mg/mL in 0.9% saline, whereas other succinateprodrugs assessed had much lower solubility. In many cases the maxsolubility of other prodrugs was lower than 100 uM. See Table 21 forexample solubility data in PBS pH7.4 for other succinate prodrugs.

TABLE 21 Solubility of example succinate prodrugs Solubility Testarticle (μM)

  Compound 1 >>100 (see text)

  A 52

Example 9—Comparison of Bioavailability of Compound 1 with OtherSuccinate Prodrugs

The cell penetrance and potential for oral bioavailability of Compound 1was tested using a standard caco-2 bioavailability in vitro assay (inbrief, confluent Caco-2 cells (L1, A. P., 1992; Grass, G. M; et al.,1992, Volpe, D. A., et al., 2001) in a 24 well Corning Costar Transwellformat were provided by In Vitro Technologies Inc. (IVT Inc., Baltimore,Md., USA). The apical chamber contained 0.15 mL Hank's balanced buffersolution (HBBS) pH 7.4, 1% DMSO, 0.1 mM Lucifer Yellow. The basalchamber contained 0.6 mL HBBS pH 7.4, 1% DMSO. Controls and tests wereincubated at 37° C. in a humidified incubator, shaken at 130 rpm for 1h. Lucifer Yellow permeates via the paracellular (between the tightjunctions) route only, a high Apparent Permeability (Papp) for LuciferYellow indicates cellular damage during assay and all such wells wererejected. Propranolol (good passive permeation with no known transportereffects) & acebutalol (poor passive permeation attenuated by activeefflux by P-glycoprotein) were used as reference compounds. Compoundswere tested in a uni- and bi-directional format by applying compound tothe apical or basal chamber (at 0.01 mM). Compounds in the apical orbasal chambers were analysed by HPLC-MS. Results were expressed asApparent Permeability, Papp, (nm/s), in comparison to other succinateprodrugs, including a number from WO2015/155231. Data is presented intable 22 below and shows that the Apical to Basolateral transfer ishighest for Compound 1, showing improved cell penetrance andbioavailability. This was confirmed by in vivo pharmacokinetic studieswhich showed Compound 1 also had a high oral bioavailability and wasbrain penetrant, unlike other prodrugs tested.

TABLE 22 Caco-2 bioavailability of Compound 1 in comparison to othersuccinate pro-drugs. P_(app)(A-B) Compound ID nm/s

  Compound 1 4.12

  A 3.13

  B 1.92

  C 1.28

  D 1.42

  E <0.21  

  F 1.45

  G 0.87

  HI 2.62

  J 1.35

  K 1.68

  L 0.16

  M 0.97

Example 10—Comparison of Thermodynamic Solubility of Different Batchesof Compound 1

Two batches of Compound 1 were generated with different crystallinity.Batch 2 had higher crystallinity than Batch 3, which is regarded ashaving a higher degree of amorphous Compound 1-Batch 2 is thereforeregarded as a more crystalline batch, whereas Batch 3 is regarded as amore amorphous batch. The methods used are discussed in this document.PBS was generated as usual (NaCl (8 g/L), KCl (0.2 g/L), disodiumhydrogen phosphate anhydrous (1.42 g/L) and potassium dihydrogenphosphate anhydrous (0.24 g/L) were added to 250 mL of deionised waterand the mixture stirred until all the solid had dissolved. The pH of thesolution was adjusted to pH 7.4 using HCl (1 M) or NaOH (1 M) asnecessary).

A sample of the more amorphous Compound 1 batch 3 was added to PBS orwater to a final concentration of 258 mg/mL and sonicated for 10minutes, then shaken for 30 minutes. Following centrifugation to removesolid material, analysis revealed a concentration of 258 mg/mL had beenreached.

A sample of the more crystalline Compound 1 batch 2 was added toHPLC-grade water (Fisher) to a final concentration of 30 mg/mL andsonicated for 10 minutes, then shaken for 30 minutes. Followingcentrifugation to remove solid material, analysis revealed aconcentration of 17 mg/mL had been reached.

A sample of the more crystalline Compound 1 batch 2 was added toHPLC-grade water (Fisher) to a final concentration of 52 mg/mL andsonicated for 20 minutes, then shaken for 1 hour. Followingcentrifugation to remove solid material, analysis revealed aconcentration of 52 mg/mL had been reached.

As can be seen from the data presented—the more amorphous material hasmuch higher kinetic solubility than the more crystalline material.

A sample of the more crystalline Compound 1 batch 2 was added toHPLC-grade water (Fisher) to a final concentration of 2000 mg/mL andsonicated for 20 minutes, then shaken for 1.5 hours. Followingcentrifugation to remove solid material, analysis revealed aconcentration of 850 mg/mL had been reached. This result show that thewater solubility of the more crystalline Compound is at least 850 mg/ml,i.e. it has a high water solubility, but the kinetic solubility ishigher for the more amorphous Compound 1.

Example 11—Comparison of Stability of Compound 1 in Purified orNon-Purified Water

A side-by-side experiment of the stability of Compound 1 in ‘purified’HPLC grade water (Fisher Scientific) and ‘non-purified’ tap water wasset up. In brief, 1 mg/mL solutions of Compound 1 were generated in‘purified’ HPLC grade water (Fisher Scientific) and ‘non-purified’ tapwater. These were incubated at room temperature for up to 10 days.Concentration and purity of Compound 1 was assessed over time by HPLC incomparison to a standard (AUC of 8.21 RT peak at 230 nm for calculatedconcentration and AUC of Compound 1 peak vs impurities for purityanalysis). Data presented is the average of two samples.

TABLE 23 Compound 1 batch 3 dissolved in tap water at 1 mg/ml, stored atroom temperature for number of days noted in table Expected CalculatedTime Conc. Conc. Purity (days) (mg/mL) (mg/mL) (%) 0 1 1.06 98.5 3 10.98 95.7 5 1 0.93 94.4 6 1 0.89 93.3 10 1 0.83 93.0

TABLE 24 Compound 1 batch 3 dissolved in HPLC grade water (FisherScientific) at 1 mg/ml, stored at room temperature for number of daysnoted in table Expected Calculated Time Conc. Conc. Purity (days)(mg/mL) (mg/mL) (%) 0 1 1.02 99.0 3 1 1.04 98.8 5 1 1.03 99.0 6 1 1.0199.0

Significant degradation of Compound 1 is noted in samples dissolved in(non-purified) tap water after storage at room temperature for 6 days,as seen by loss in purity and assay. This is not observed for thesamples dissolved in (purified) HPLC grade water (Fisher Scientific).Similar data was seen for two independent samples of different batchesof Compound 1.

Example 12—HP-Cyclodextrin Formulation of Compound 1

Excipient Preparation

Kleptose hydroxypropyl β-Cyclodextrin (25% w/v), sodium dihydrogenphosphate anhydrous (0.048% w/v), disodium hydrogen phosphate dihydrate(0.295% w/v) and calcium disodium EDTA (0.5% w/v) were added to 100 mLdeionised water. The mixture was sonicated for 20 mins then stirreduntil all the solid had dissolved then adjusted to pH 7.4 with HCl (1M).

Formulation Protocol

-   -   1. Weigh out the required amount of Compound 1 then add the        required amount of prepared excipient to the solid Compound 1 to        give the required mg/ml concentration of Compound 1 (maximum        tested 25 mg/ml). For example, for a 20 mg/ml formulation of        Compound 1, weigh out 20 mg of Compound 1 and add 1 ml of the        prepared excipient.    -   2. Sonicate the solution for 10 mins and then shake for 20 mins        to ensure that Compound 1 is fully dissolved.    -   3. The solution can be centrifuged (13000 rpm, 10 mins) to        remove any particulates if required.    -   4. The solution can be sterile filtered if required.

Example 13—Infusion of Compound 1 Delivers Succinate, IncreasesSuccinate Metabolism in Pigs and Reduces Blood Lactate Concentrations

Infusion of Compound 1 increases plasma succinate levels, increasesmetabolism of succinate to fumarate in tissues and decreases bloodlactate concentrations. See FIG. 7.

Yorkshire landrace hybrid pigs were anaesthetised and implanted withvenous catheters for infusions of Compound 1 or vehicle (PBS) andcollection of blood samples. Two animals received an escalating dose ofCompound 1 (2-6 mg/kg/min) over a period of 2.5 hours, where the dosewas increased by 1 mg/kg/min every 30 minutes. One animal was infused ata constant rate of 2 mg/kg/min. The control animal was infused with PBS.Blood samples were taken with 30 min intervals and plasma was separatedby centrifugation. Plasma and tissue samples were stored frozen andlater analysed for succinate in an LC/MS method using a Thermo VanquishUPLC+Thermo Quantis triple quadrupole MS instrument, an Acquity UPLC HSSC18 (100×2.1 mm, 1.8 μm) column with guard filter and gradient elution;A=0.1% Formic acid, B=Acetonitrile. [13C]-labelled succinate was used asinternal standard.

Plasma succinate concentrations increased proportional to the time ofCompound 1 infusion (FIG. 7A) demonstrating release of succinate fromCompound 1. At the end of the study, fumarate, the primary metabolite ofsuccinate in the TCA cycle was higher in tissues of the animal receivingCompound 1 than in the vehicle animal particularly in tissues with highmetabolic activity such as retina, brain and heart. The data thereforesuggest that Compound 1 delivers metabolizable succinate to thesetissues and has the ability to pass the blood brain barrier.

Blood lactate data combined from three animals were expressed aspercentage of the initial value and plotted as a function of thecumulative dose at the time of sampling (FIG. 7C).

Lactate decreased relative to initial values after intravenous infusionof Compound 1 suggesting that Compound 1 delivers succinate to complex 2and increases the supply of electrons to the mitochondrial electrontransport chain to increase ATP production and decrease the need forglycolytic conversion of pyruvate to lactate.

Example 14—Pig Model of Rotenone Induced Mitochondrial Complex 1Dysfunction

Infusion of Compound 1 restores rotenone depleted succinate levels inthe organs and decreases rotenone induced lactate in the brain. See FIG.8.

To study the effect of rotenone-mediated inhibition of complex 1,Yorkshire landrace hybrid pigs were anaesthetised and implanted withvenous catheters for simultaneous infusion of rotenone and Compound 1 orvehicle (PBS). Rotenone (7.1 mg/hr) was infused during 1.5 hours.Compound 1 was infused at a constant rate of 2 mg/kg/min over a periodof 2.5 hours. The control animal was infused with PBS. Blood sampleswere taken with 30 min intervals and plasma was separated bycentrifugation. Microdialysates were collected by inserting amicrodialysis probe into the striatum of the brain and analysed forlactate using an ISCUS instrument (MDialysis). At the end of infusion,the animal was euthanised and terminal blood and organ samples werecollected. Plasma and tissue samples were stored frozen and lateranalysed for succinate in an LC/MS method using a Thermo VanquishUPLC+Thermo Quantis triple quadrupole MS instrument, an Acquity UPLC HSSC18 (100×2.1 mm, 1.8 μm) column with guard filter and gradient elution;A=0.1% Formic acid, B=Acetonitrile. [13C]-labelled succinate was used asinternal standard. Lactate data were expressed as percentage of thebaseline value obtained before start of rotenone infusion.

Rotenone infusion decrease tissue concentrations of succinate to a levelbelow quantification (<2 μM) indicating an increased utilisation ofsuccinate to compensate for the decrease in electron transfer fromcomplex 1. Administration of Compound 1 restored tissue succinateconcentrations to detectable levels suggesting that the delivery ofsuccinate by Compound 1 exceeded the increased succinate utilisationcaused by complex 1 inhibition. Furthermore, administration of Compound1 counteracted a rotenone induced increase in brain lactate, confirminga decreased need for glycolytic conversion of pyruvate to lactate bydelivery of succinate to the brain.

Example 15—Mouse Genetic Ndufs4 Knock-Out Model of Complex 1 Dysfunction

Administration of Compound 1 in the drinking water (1 mg/mL) fromweaning (day 21) results in transiently increased body weight and atrend of prolonged survival in the high concentration group. See FIG. 9.

C57BL/6 mice with a genetic ablation of the complex I gene Ndufs4(Quintana et al., Proceedings of the National Academy of Sciences. 107,24 (2010), 10996-11001) were given Compound 1 (1 mg/mL) in the drinkingwater or plain drinking water from weaning. Body weight development wasmonitored every 10 days (FIG. 9A) and animal health was monitored daily(FIG. 9B). Compound 1 increased weigh gain (p<0.05) during the first 10days and increased the survival rate (p=0.0697). The results suggestthat a therapeutic benefit of succinate supplementation in the form ofCompound 1 can be achieved in a genetic model of mitochondrial complex Idysfunction I with features of Leigh syndrome.

Example 16—Rat Model of Rotenone Induced Motor Dysfunction and LacticAcidosis

Compound 1 administered in the drinking water prevents motordysfunctions and reduces blood lactate concentrations. See FIG. 10

A rotenone induced rat Parkinson disease model (Cannon et al., NeurobiolDis. 2009 May; 34(2):279-90) was used to study the effect of oraladministration of Compound 1 on motoric and metabolic dysfunctionscaused by complex 1 inhibition.

Twelve-week old Lewis rats (6 animals per group) received dailyintraperitoneal injections of rotenone (0.25-0.75 mg/kg) for 4 days.Compound 1 was dissolved in the drinking water at a concentration of0.25 and 0.75 mg/mL. Functional tests and lactate measurements wereperformed day 4. Rearing was measured by placing animals in a clearglass cylinder (height=30 cm; diameter=18 cm) during five minutes. To beclassified as rearing, the forelimbs should be raised above shoulderlevel and make contact with the cylinder wall with either one or bothforelimbs.

Postural instability was measured on a table-top covered with P-120sandpaper, marked with lines and numbers every centimetre (see below).The animal was held in a vertical position (“wheelbarrow”-likeposition), at a nearly 90° angle to the surface with one forelimb gentlyrestrained against the animal's torso. The centre of gravity of theanimal was then shifted forward over the single planted forelimb totrigger two “catch-up” steps, to regain its balance. The change inposition of the nose was recorded as the distance that triggered acatch-up step in the unrestrained forelimb. The experiment was repeatedthree times for each forelimb and an average of for both forelimbs wascalculated. Blood lactate (FIG. 100) was measured in a VetScan iSTAT-1Analyser.

Rotenone treatment resulted in decrease rearing activity. Administrationof Compound 1 (0.75 mg/mL) in the drinking water resulted in asignificant increase in rearings (FIG. 10A) and postural instability(FIG. 10B) compared to treated animals receiving water. The datatherefore suggest that Compound 1 is orally bioavailable and is able toameliorate motoric dysfunctions caused by mitochondrial complex 1dysfunction. Blood lactate concentrations were increased significantlyin animals treated with rotenone. There was a trend of decreasinglactate concentrations in blood from the low concentration (0.25 mg/mL)of Compound 1 in the drinking water to the high concentration (0.75mg/mL). The data suggest that succinate delivered from Compound 1 canachieve metabolic compensation at the level of glycolysis and conversionof pyruvate to lactate when delivered via the oral route by intermittentadministration in the drinking water and implicates suitability as anoral treatment.

Example 17—Succinate Release Data

In brief, a stock of Compound 1 Batch 12 was prepared in 50/50 DMSO/MeCN(200 mM, ×200). This mixture was then diluted 1 in 10 into microsomebuffer (20 mM, ×20) consisting of K₂HPO₄ (Sigma Aldrich, 13.9 g/L,anhydrous), KH₂PO₄ (Sigma Aldrich, 2.72 g/L, anhydrous), MgCl₂.6H₂O(Fisher, 1.02 g/L) and EDTA (Sigma Aldrich, 0.375 g/L) dissolved in HPLCgrade water. A 200 mM malonic acid (Sigma Aldrich) stock was thenprepared in microsome buffer (200 mM, ×20). A 20 mM NADPH stock was alsoprepared in microsome buffer (20 mM, ×10). A stock of microsomes(Sekisui XenoTech, 0.625 mg/ml) was prepared in a 7 ml vial. Sampleswere prepared for each timepoint (T=0, 5, 15, 60 mins) as follows: 80 μLmicrosome stock (0.5 mg/mL final concentration), 5 μL of Compound stock(2 mM final concentration), 5 μL of malonate stock (10 mM finalconcentration). The T=0 sample was quenched by adding 100 μL MeOH.Reaction of all timepoints was then initiated with addition of 10 μLNADPH stock (2 mM final concentration). At each timepoint, the reactionwas terminated by the addition of 100 μL MeOH. Samples were shaken for 1min, placed on ice for 10 min, then centrifuged at 3000 rpm for 10minutes. The supernatant was then analysed by LCMS.

TABLE 25 Species detected by LCMS Succinic Mono-methyl Time Compound 1acid succinate (min) (mM) (mM) (mM) 0 0.934 0.058 0.088 5 0.688 0.1360.161 15 0.388 0.285 0.205 60 0.080 0.599 0.308

As can be seen from the data, Compound 1 releases succinic acid overtime when incubated with microsomes.

Example 18—Mineral Instability

Compound 1 Batch 14 (2-3 mg) was dissolved in water for injection atroom temperature (WFI) (1 mg/ml) containing different sources ofminerals commonly found in tap water (with different accompanyingcounter ions to be able to distinguish differences between the effectsof cation or anion) and samples were taken at different time intervalsfor HPLC analysis.

Mineral sources used in the form of inorganic salts:

CuCl₂, CaCl₂), NiSO₄, CoCl₂, NH₄Cl, MnCl₂, NaF, NaNO₃, CuSO₄, Ca(NO₃)₂,AlSO₄, CaCO₃, (NH₄)₂CO₃.

TABLE 26 Percentage (%) of Compound 1 remaining when compared to the T =0 timepoint for Time each different salt added to HPLC water (days) WFlCuCl₂ CaCl₂ NiSO₄ CoCl₂ NH₄Cl MnCl₂ 0 100 100 100 100 100 100 100 7 98.799.5 100 98.4 101 101 98.9 14 97.7 98.3 97.9 99.1 101 99.8 99.2Percentage (%) of Compound 1 remaining when compared to the T = 0timepoint for each different salt added to HPLC water Time (NH₄)₂ (days)NaF NaNO₃ CuSO₄ Ca(NO₃)₂ AlSO₄ CaCO₃ CO₃ 0 100 100 100 100 100 100 100 7101 99.2 100 99.4 99.8 79.5 91.3 14 100 99.4 99.4 98.9 100 66.0 87.4

As can be seen from the data, Compound 1 degrades more rapidly when inaqueous solution with carbonate ions.

Example 19—Carbonate Instability

Compound 1 Batch 14 (2-3 mg) was dissolved in HPLC grade water at roomtemperature (1 mg/ml) containing different sources of calcium andcarbonate ions (two concentrations for each source, with the pHrecorded) and samples were taken at different time intervals for HPLCanalysis.

Calcium and carbonate sources used in the form of inorganic salts:

CaCl₂.Ca(NO₃)₂, CaCO₃, (NH₄)₂CO₃

TABLE 27 Percentage (%) of Compound 1 remaining when compared to the T =0 timepoint for each different mineral added to HPLC water HPLC waterCaCO₃ CaCO₃ CaCl₂ CaCl₂ Time (control, (3.4 mM, (0.34 mM, (3.4 mM, (0.34mM, (days) pH 6.5) pH 7.6) pH 7.6) pH 7.5) pH 7.4) 0 100 100 100 100 1003 94.3 93.6 93.8 101 98.4 17 91.4 49.5 50.4 96.7 95.1 Percentage (%) ofCompound 1 remaining when compared to the T = 0 timepoint for eachdifferent mineral added to HPLC water Ca(NO₃)₂ Ca(NO₃)₂ (NH₄)₂CO₃(NH₄)₂CO₃ Time (3.4 mM, (0.34 mM, (3.4 mM, (3.4 mM, (days) pH 7.2) pH7.3) pH 9.3) pH 8.9) 0 100 100 100 100 3 100 101 98.9 99.6 17 97.1 97.557.5 85.5

As can be seen from the data, Compound 1 degrades more rapidly when inaqueous solution with carbonate ions.

Example 19—Carbonate Concentration Dependence

Compound 1 Batch 14 (2-3 mg) was dissolved in HPLC grade water (1 mg/ml)containing different concentrations of calcium carbonate and sampleswere taken at different time intervals for HPLC analysis.

TABLE 28 Percentage (%) of Compound 1 remaining when compared to the T =0 timepoint for each different mineral added to HPLC water Time CaCO₃CaCO₃ CaCO₃ CaCO₃ CaCO₃ (days) (0 mM) (0.425 mM) (0.85 mM) (1.7 mM) (3.4mM) 0 100 100 100 100 100 7 98.7 98.1 97.8 93.8 83.4 14 98.6 98.4 97.190.8 71.4

Example 20—Kinetic Solubility

Solid Compound 1 Batches s3, s12-17 (˜80 mg) were added to wells of aflat clear bottom 96 well plate. PBS at 5° C. was added to give a 460mg/ml final concentration of 01-354 for all batches. After addition ofthe PBS, the plate was agitated for 10 s then immediately analysed forturbidity at 620 nm in real time for 3 mins by an Epoch MicroplateSpectrophotometer (BioTek). A rate constant for the dissolution wascalculated from the exponential decay fit of the recorded data.

TABLE 29 Compound 1 Rate constants, Batch No. k (s⁻¹) 3 0.014 12 0.02213 0.013 14 0.011 15 0.074 16 0.055 17 0.111

General Method for Assessing Crystallinity

X-ray powder diffractions studies were conducted using a Bruker AXS D8discover HTS.

Anode: Cu anode at 40 kV and 4 mV; Göbel mirror and line optics.

Detector: linear detector (LYNXEYE XE) with receiver slit of 2.95°.

Measurement: scan range 2-45° 2θ, 1 s/step, 0.005°/step

Data collection software: Diffrac. Commander v7.3.3.0.0

Data analysis software: Diffrac Eva v4.2.1

No background correction or smoothing was applied. Data are reported aspeak 2θ angle and intensity. In order to determine the extent ofcrystallinity the combined area for all defined peaks was divided by thetotal area under curve and expressed as a percentage.

Peak List

The following list details the peaks returned after XRPD on a number ofcrystalline batches. Peaks underlined are common to most batches. Thosewhere there is an asterisk next to the angle may be peaks more common orunique to polymorphic forms.

Peak list NV354-s3—batch 3

Rel. Angle d Value Intensity Intensity 9.4 9.389 54 1.4% 10.9 8.136 453 12.1%  11.1* 7.997 1270  33.7%  11.4 7.777 110  2.9% 12.9 6.870 208 5.5% 13.1 6.776 1240  32.9%  13.1 6.728 120  3.2% 13.8 6.420 36 1.0%14.5 6.104 60 1.6% 14.9 5.939 406  10.8%  15.6 5.659 63 1.7% 16.2 5.476179  4.8% 16.9* 5.246 89 2.4% 17.9 4.941 89 2.4% 18.5 4.785 49 1.3% 19.24.624 176  4.7% 19.6 4.528 226  6.0% 19.7 4.499 214  5.7% 20.1 4.418111  3.0% 21.4 4.143 831  22.1%  21.7 4.100 95 2.5% 22.2 3.994 163  4.3%22.7 3.919 698  18.6%  22.8 3.893 1410  37.4%  23.1 3.847 2760  73.4% 23.3 3.813 307  8.2% 24.0 3.711 3760  100.0%  24.4 3.640 58 1.5% 24.83.591 602  16.0%  25.2 3.538 78 2.1% 25.5 3.487 283  7.5% 26.1 3.4091340  35.5%  27.2 3.282 53 1.4% 27.7 3.221 76 2.0% 28.0 3.185 58 1.5%28.5 3.128 48 1.3% 29.9 2.989 69 1.8% 30.2 2.956 115  3.1% 30.8 2.901 892.4% 30.9 2.890 73 1.9% 31.3 2.857 64 1.7% 31.6 2.829 29 0.8% 32.2 2.77642 1.1% 32.8 2.730 27 0.7% 33.0 2.711 42 1.1% 33.2 2.694 59 1.6% 35.02.560 73 1.9% 35.4 2.536 37 1.0% 36.6 2.456 86 2.3% 38.6 2.333 49 1.3%40.1 2.248 52 1.4% 41.6 2.169 58 1.5% 44.7 2.026 86 2.3% 44.8 2.022 571.5%

Peak list NV354-s12—batch 18

Rel. Angle d Value Intensity Intensity 5.6 15.805 68 3.3% 9.4 9.390 994.9% 10.9 8.141 233 11.5% 11.1 7.949 365 18.0% 11.2 7.883 635 31.3% 11.47.744 252 12.4% 12.9 6.848 183 9.0% 13.2 6.710 103 5.1% 13.8 6.432 1155.7% 14.9 5.941 446 22.0% 15.7 5.657 91 4.5% 16.2 5.484 312 15.4% 16.95.243 288 14.2% 17.3 5.119 107 5.3% 18.1 4.902 87 4.3% 18.6 4.779 603.0% 19.3 4.607 274 13.5% 19.3 4.594 154 7.6% 19.7 4.506 164 8.1% 20.14.411 505 25.0% 21.4 4.148 1030 51.1% 21.6 4.110 160 7.9% 22.2 4.001 39519.5% 22.7 3.919 2030 100.0% 22.8 3.892 1960 96.6% 23.0 3.865 1990 98.3%23.2 3.824 204 10.1% 24.0 3.709 90 4.5% 24.5 3.637 122 6.0% 24.8 3.593226 11.1% 25.1 3.539 227 11.2% 25.5 3.487 126 6.2% 26.1 3.407 151 7.4%27.8 3.210 196 9.7% 28.5 3.124 53 2.6% 32.8 2.728 41 2.0% 35.0 2.560 643.2% 37.7 2.386 75 3.7% 41.6 2.169 62 3.0% 43.0 2.104 26 1.3%

Peak list NV354-s13—batch 13

Rel. Angle d Value Intensity Intensity 7.9 11.190 39 2.3% 9.4 9.405 1186.9% 10.9 8.136 668 39.3% 11.1 7.963 408 24.0% 11.2 7.884 294 17.3% 11.47.743 179 10.5% 12.9 6.858 209 12.3% 13.2 6.709 103 6.1% 13.8 6.432 261.6% 14.5 6.099 71 4.2% 14.9 5.954 474 27.9% 15.6 5.659 155 9.1% 16.25.472 326 19.2% 16.9 5.250 134 7.9% 17.4 5.102 44 2.6% 18.1 4.906 895.2% 19.0 4.677 57 3.3% 19.2 4.619 269 15.8% 19.4 4.577 180 10.6% 19.74.505 162 9.5% 20.1 4.414 392 23.0% 21.4 4.145 536 31.5% 21.6 4.106 794.7% 22.3 3.990 396 23.3% 22.7 3.913 898 52.7% 22.9 3.887 1700 100.0%23.1 3.855 1380 80.9% 23.3 3.812 336 19.8% 24.0 3.711 84 4.9% 24.6 3.620131 7.7% 24.8 3.590 371 21.8% 25.2 3.536 152 8.9% 25.5 3.487 94 5.5%26.1 3.407 195 11.4% 27.2 3.280 71 4.2% 27.8 3.210 100 5.9% 28.0 3.18590 5.3% 29.6 3.015 47 2.8% 30.2 2.959 34 2.0% 30.8 2.902 72 4.2% 31.32.858 37 2.2% 31.6 2.827 27 1.6% 32.0 2.795 68 4.0% 32.8 2.730 79 4.6%34.2 2.620 65 3.8% 34.4 2.602 52 3.0% 35.1 2.555 63 3.7% 35.4 2.535 663.9% 37.7 2.384 44 2.6% 40.1 2.246 35 2.1% 41.6 2.170 68 4.0% 44.2 2.04927 1.6%

Peak list NV354-s14—batch 14

Rel. Angle d Value Intensity Intensity 7.9 11.145 111 5.5% 9.4 9.370 1326.5% 10.8 8.175 130 6.4% 10.9 8.132 74 3.7% 11.1 7.992 343 16.9% 11.47.750 184 9.0% 12.9 6.841 73 3.6% 13.2 6.713 169 8.3% 14.9 5.935 1135.6% 15.6 5.663 80 3.9% 16.2 5.484 161 7.9% 16.9 5.248 47 2.3% 18.04.937 33 1.6% 18.6 4.777 52 2.6% 19.4 4.564 220 10.9% 19.7 4.502 1959.6% 20.1 4.421 46 2.3% 21.4 4.143 1150 56.5% 22.2 4.000 355 17.5% 22.73.921 2030 100.0% 22.9 3.888 509 25.1% 23.0 3.857 812 40.0% 24.5 3.624173 8.5% 24.8 3.592 40 2.0% 25.2 3.537 89 4.4% 25.5 3.486 346 17.0% 26.03.427 122 6.0% 26.2 3.397 169 8.3% 27.1 3.282 63 3.1% 27.8 3.208 26212.9% 28.0 3.182 57 2.8% 28.6 3.123 43 2.1% 28.7 3.113 52 2.6% 29.63.012 30 1.5% 30.3 2.951 82 4.0% 30.8 2.901 55 2.7% 31.2 2.864 31 1.5%32.2 2.775 22 1.1% 33.0 2.711 55 2.7% 34.2 2.618 83 4.1% 34.8 2.577 442.2% 35.1 2.556 57 2.8% 36.2 2.483 167 8.2% 37.3 2.408 69 3.4% 37.72.386 44 2.2% 39.8 2.266 26 1.3% 41.6 2.169 83 4.1% 41.7 2.164 45 2.2%

Peak list NV354-s15—batch 19

Rel. Angle d Value Intensity Intensity 5.6 15.862 72 4.6% 7.9 11.170 523.3% 9.4 9.389 134 8.5% 10.8 8.151 388 24.6% 11.1 7.976 412 26.2% 11.27.907 400 25.4% 11.4 7.765 121 7.7% 12.9 6.846 108 6.8% 13.2 6.714 21113.4% 13.7 6.440 41 2.6% 14.9 5.936 456 29.0% 15.6 5.660 134 8.5% 16.25.478 312 19.8% 16.9 5.252 122 7.7% 18.1 4.906 49 3.1% 19.0 4.673 583.7% 19.2 4.614 96 6.1% 19.7 4.506 170 10.8% 20.1 4.413 162 10.3% 21.44.143 943 59.9% 21.6 4.104 306 19.5% 22.2 3.997 318 20.2% 22.8 3.8941400 88.7% 23.1 3.854 1570 100.0% 23.3 3.813 488 31.0% 23.9 3.716 815.1% 24.5 3.628 74 4.7% 24.5 3.627 109 6.9% 24.8 3.592 139 8.8% 25.23.535 147 9.3% 25.6 3.482 146 9.3% 26.1 3.406 139 8.8% 27.2 3.281 523.3% 27.8 3.211 122 7.8% 29.6 3.012 34 2.2% 30.8 2.900 62 3.9% 33.02.715 25 1.6% 41.6 2.170 38 2.4%

Peak list NV354-s16—batch 16

Rel. Angle d Value Intensity Intensity 5.6 15.858 91 4.2% 7.9 11.148 632.9% 9.4 9.395 138 6.3% 10.9 8.134 401 18.4% 11.1 7.945 354 16.3% 11.27.891 514 23.6% 11.4 7.748 167 7.7% 12.9 6.852 100 4.6% 13.2 6.709 1758.0% 13.8 6.424 72 3.3% 14.5 6.091 54 2.5% 14.9 5.943 370 17.0% 15.75.656 151 7.0% 16.2 5.479 233 10.7% 16.9 5.244 248 11.4% 17.4 5.106 311.4% 18.1 4.895 106 4.9% 18.6 4.777 43 2.0% 19.2 4.613 284 13.1% 19.44.583 214 9.8% 19.7 4.507 179 8.2% 20.1 4.413 274 12.6% 21.4 4.147 62228.6% 21.6 4.102 210 9.7% 22.2 3.995 293 13.5% 22.7 3.921 834 38.3% 22.83.891 1490 68.2% 23.1 3.855 2180 100.0% 23.3 3.813 279 12.8% 24.0 3.71187 4.0% 24.5 3.627 100 4.6% 24.8 3.592 266 12.2% 25.2 3.533 180 8.3%25.5 3.489 122 5.6% 26.2 3.403 162 7.4% 27.1 3.283 36 1.6% 27.8 3.207173 7.9% 28.0 3.184 49 2.2% 28.5 3.125 34 1.6% 29.6 3.015 33 1.5% 30.82.905 82 3.8% 31.6 2.829 44 2.0% 32.0 2.791 24 1.1% 32.8 2.725 42 1.9%34.2 2.618 71 3.3% 35.0 2.559 61 2.8% 35.4 2.537 28 1.3% 36.1 2.484 351.6% 37.7 2.387 48 2.2% 40.2 2.244 47 2.2% 41.6 2.170 98 4.5%

Peak list NV354-s17—batch 17

Rel. Angle d Value Intensity Intensity 7.9 11.171 108 2.6% 9.4 9.422 771.8% 10.8 8.158 297 7.1% 11.1 7.984 379 9.0% 11.2 7.873 790 18.9% 11.47.753 134 3.2% 12.8 6.892 114 2.7% 12.9 6.845 185 4.4% 13.2 6.715 1794.3% 13.8 6.420 77 1.8% 14.9 5.931 349 8.3% 15.7 5.649 225 5.4% 16.25.462 184 4.4% 16.9 5.248 303 7.2% 17.9 4.948 52 1.3% 18.5 4.785 40 0.9%19.2 4.625 183 4.4% 19.3 4.586 263 6.3% 19.7 4.500 307 7.3% 20.1 4.416220 5.3% 21.4 4.148 501 12.0% 21.6 4.105 145 3.5% 22.2 3.998 1100 26.3%22.8 3.890 1700 40.6% 23.1 3.853 2000 47.7% 23.2 3.832 4190 100.0% 24.63.616 264 6.3% 24.8 3.590 771 18.4% 25.2 3.535 230 5.5% 25.5 3.489 1273.0% 26.1 3.410 242 5.8% 27.8 3.207 285 6.8% 29.6 3.015 24 0.6% 30.82.901 151 3.6% 30.9 2.896 155 3.7% 31.2 2.861 314 7.5% 31.6 2.830 1603.8% 33.1 2.707 38 0.9% 33.7 2.658 164 3.9% 34.2 2.621 62 1.5% 35.02.561 103 2.5% 38.6 2.331 95 2.3% 38.9 2.311 44 1.0% 40.7 2.216 77 1.8%41.6 2.171 48 1.1% 42.4 2.129 34 0.8%

1. Isolated Methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate(Compound 1) in solid form, having a purity of at least 80% w/w, atleast 90% w/w, at least 95% w/w, at least 97% w/w, at least 98% w/w orat least 99% w/w.
 2. The isolated Compound 1 according to claim 1, whichis in the form of the free form or a salt, hydrate, solvate or complexthereof.
 3. The isolated Compound 1 according to claim 1 having amelting point or a melting range in a range of from 35 to 55° C.
 4. Theisolated Compound 1 according to claim 1, which is a crystalline productor an amorphous product, or a mixture thereof.
 5. The isolated Compound1 according to claim 1 having an aqueous solubility at room temperatureof at least 300 mg/mL.
 6. The isolated Compound 1 according to claim 5,wherein the aqueous solubility at room temperature is in a range of from300 mg/mL to 900 mg/ml.
 7. The isolated Compound 1 according to claim 1having a crystallinity in a range of from 0 to 100% such as from 50 to100%.
 8. The isolated Compound 1 according to claim 1 having a kineticaqueous solubility corresponding to a rate constant in a range of from0.005 to 0.2 s⁻¹.
 9. The isolated Compound 1 according to claim 1 havingan X-ray powder diffraction pattern with one or more signals at 21.4,22.2, 22.8, 23.1 and 23.3 (±0.2 degrees, 2-theta values).
 10. Theisolated Compound 1 according to claim 1 having an X-ray powerdiffraction pattern with one or more signals at 10.9, 13.1, 14.9, 16.2,20.1, 24.0, 24.8, 26.1, (±0.2 degrees, 2-theta values).
 11. (canceled)12. (canceled)
 13. A method for treating or preventing a metabolicdisease, a disease of mitochondrial dysfunction, a disease related tomitochondrial dysfunction, a mitochondrial disorder, mitochondrialenergy deficiency, drug-induced mitochondrial side effects, cancer,diabetes, traumatic brain injury, acute liver injury, or atrialfibrillation, said method comprising administering the isolated Compound1 according to claim 1 to a subject in need thereof.
 14. The methodaccording to claim 13 for treating or preventing metabolic dysfunction.15. The method according to claim 13 for treating or preventing LeighSyndrome, LHON, MELAS, MERRF (myoclonic epilepsy with ragged redfibers), or other diseases/conditions relating to mitochondrial ComplexI defects.
 16. (canceled)
 17. Cosmeceutical comprising the isolatedCompound 1 according to claim
 1. 18. Nutricosmetics comprising theisolated Compound 1 according to claim
 1. 19. Energy drink comprisingthe isolated Compound 1 according to claim
 1. 20. Pharmaceuticalcomposition comprising isolated Compound 1 according to claim
 1. 21. Thecompound according to claim 1 in the form of its Form I crystallinepolymorph having an X-ray powder diffraction pattern with signals at11.2, 16.9 (±0.2 degrees, 2-theta values). 22-25. (canceled)